Increasing bioavailability of carotenoids

ABSTRACT

A method of increasing a fraction of free carotenoids in a source of carotenoids in which at least some of the carotenoids are fatty acid esterified carotenoids is disclosed. The method is effected by contacting the source of carotenoids with an effective amount of an esterase under conditions effective in deesterifying the fatty acid esterified carotenoids, thereby increasing the fraction of free carotenoids in the source of carotenoids.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.10/661,606, filed on Sep. 15, 2003, which is a Continuation-in-Part(CIP) of PCT Application No. PCT/IL02/00398, filed on May 21, 2002,which claims priority from U.S. patent application Ser. No. 09/915,527,filed on Jul. 27, 2001, now abandoned, and U.S. Provisional PatentApplication No. 60/292,953, filed on May 24, 2001, now expired. Thecontents of the above Applications are incorporated herewith byreference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to a novel method of increasing thebioavailability of carotenoids. More particularly, the present inventionrelates to methods of extracting oleoresin, increasing the content offree carotenoids in sources of carotenoids rich in fatty acid esterifiedcarotenoids, red pepper in particular. The present invention furtherrelates to the extraction of free carotenoids from the sources ofcarotenoids rich in fatty acid esterified carotenoids and to food andfeed additives that comprise free carotenoids.

Carotenoids, Chemistry and Biochemistry:

The carotenoids are isoprenoid compounds, with an extensive conjugateddouble bond system, and are biosynthesized from acetyl coenzyme-A viamevalonic acid as a branch of the great isoprenoid or terpenoid pathway(Britton, 1996). They are divided into two main classes; carotenes[acyclic (lycopene) and cyclic (β-carotene)], and xanthophylls (e.g.,capsanthin). In contrast to carotenes, which are pure polyenehydrocarbons, xanthophylls also contain hydroxy, epoxy and keto groups.Only plants, and microorganisms synthesize carotenoids, however they arereach by feed and food animal or human tissues, which have the abilityto absorb, modify and store these compounds (Goodwin; 1980).

Of the over 640 carotenoids found in nature, about 20 are present in atypical human diet. Of these carotenoids, only 14 and some of theirmetabolites have been identified in blood and tissues (Gerster, 1997;Khackick et al., 1995; Oshima, et al., 1997).

As part of the light-harvesting antenna, carotenoids can absorb photonsand transfer the energy to chlorophyll, thus assisting in the harvestingof light in the range of 450-570 nm [see, Cogdell R J and Frank H A(1987) How carotenoids function in photosynthetic bacteria. BiochimBiophys Acta 895: 63-79; Cogdell R (1988) The function of pigments inchloroplasts. In: Goodwin T W (ed) Plant Pigments, pp 183-255. AcademicPress, London; Frank H A, Violette C A, Trautman J K, Shreve A P, OwensT G and Albrecht A C (1991) Carotenoids in photosynthesis: structure andphotochemistry. Pure Appl Chem 63: 109-114; Frank H A, Farhoosh R,Decoster B and Christensen R L (1992) Molecular features that controlthe efficiency of carotenoid-to-chlorophyll energy transfer inphotosynthesis. In: Murata N (ed) Research in Photosynthesis, Vol I, pp125-128. Kluwer, Dordrecht; and, Cogdell R J and Gardiner A T (1993)Functions of carotenoids in photosynthesis. Meth Enzymol 214: 185-193].Although carotenoids are integral constituents of the protein-pigmentcomplexes of the light-harvesting antennae in photosynthetic organisms,they are also important components of the photosynthetic reactioncenters.

Most of the total carotenoids is located in the light harvesting complexII [Bassi R, Pineaw B, Dainese P and Marquartt J (1993) Carotenoidbinding proteins of photosystem II. Eur J Biochem 212: 297-302]. Theidentities of the photosynthetically active carotenoproteins and theirprecise location in light-harvesting systems are not known. Carotenoidsin photochemically active chlorophyll-protein complexes of thethermophilic cyanobacterium Synechococcus sp. were investigated bylinear dichroism spectroscopy of oriented samples [see, Breton J andKato S (1987) Orientation of the pigments in photosystem II:low-temperature linear-dichroism study of a core particle and of itschlorophyl]-protein subunits isolated from Synechococcus sp. BiochimBiophys Acta 892: 99-107]. These complexes contained mainly a β-carotenepool absorbing around 505 and 470 nm, which is oriented close to themembrane plane. In photochemically inactive chlorophyll-proteincomplexes, the α-carotene absorbs around 495 and 465 nm, and themolecules are oriented perpendicular to the membrane plane.

Evidence that carotenoids are associated with cyanobacterial photosystem(PS) II has been described [see, Suzuki R and Fujita Y (1977) Carotenoidphotobleaching induced by the action of photosynthetic reaction centerII: DCMU sensitivity. Plant Cell Physiol 18: 625-631; and, Newman P Jand Sherman L A (1978) Isolation and characterization of photosystem Iand II membrane particles from the blue-green alga Synechococcuscedrorum. Biochim Biophys Acta 503: 343-361]. There are two β-carotenemolecules in the reaction center core of PS II [see, Ohno T, Satoh K andKatoh S (1986) Chemical composition of purified oxygen-evolvingcomplexes from the thermophilic cyanobacterium Synechococcus sp. BiochimBiophys Acta 852: 1-8; Gounaris K, Chapman D J and Barber J (1989)Isolation and characterization of a D1/D2/cytochrome b-559 complex fromSynechocystis PCC6803. Biochim Biophys Acta 973: 296-301; and, Newell RW, van Amerongen H, Barber J and van Grondelle R (1993) Spectroscopiccharacterization of the reaction center of photosystem II usingpolarized light: Evidence for β-carotene excitors in PS II reactioncenters. Biochim Biophys Acta 1057: 232-238] whose exact function(s) isstill obscure [reviewed by Satoh K (1992) Structure and function of PSII reaction center. In: Murata N (ed) Research in Photosynthesis, Vol.II, pp. 3-12. Kluwer, Dordrecht]. It was demonstrated that these twocoupled α-carotene molecules protect chlorophyll P680 from photodamagein isolated PS II reaction centers [see, De Las Rivas J, Telfer A andBarber J (1993) 2-coupled β-carotene molecules protect P680 fromphotodamage in isolated PS II reaction centers. Biochim. Biophys. Acta1142: 155-164], and this may be related to the protection againstdegradation of the D1 subunit of PS II [see, Sandmann G (1993) Genes andenzymes involved in the desaturation reactions from phytoene tolycopene. (abstract), 10th International Symposium on Carotenoids,Trondheim CL1-2]. The light-harvesting pigments of a highly purified,oxygen-evolving PS II complex of the thermophilic cyanobacteriumSynechococcus sp. consists of 50 chlorophyll a and 7 β-carotene, but noxanthophyll, molecules [see, Ohno T, Satoh K and Katoh S (1986) Chemicalcomposition of purified oxygen-evolving complexes from the thermophiliccyanobacterium Synechococcus sp. Biochim Biophys Acta 852: 1-8].β-carotene was shown to play a role in the assembly of an active PS IIin green algae [see, Humbeck K, Romer S and Senger H (1989) Evidence forthe essential role of carotenoids in the assembly of an active PS II.Planta 179: 242-250].

Isolated complexes of PS I from Phormidium luridum, which contained 40chlorophylls per P700, contained an average of 1.3 molecules ofβ-carotene [see, Thornber J P, Alberte R S, Hunter F A, Shiozawa J A andKan K S (1976) The organization of chlorophyll in the plantphotosynthetic unit. Brookhaven Symp Biology 28: 132-148]. In apreparation of PS I particles from Synechococcus sp. strain PCC 6301,which contained 130±5 molecules of antenna chlorophylls per P700, 16molecules of carotenoids were detected [see, Lundell D J, Glazer A N,Melis A and Malkin R (1985) Characterization of a cyanobacterialphotosystem I complex. J Biol Chem 260: 646-654]. A substantial contentof β-carotene and the xanthophylls cryptoxanthin and isocryptoxanthinwere detected in PS I pigment-protein complexes of the thermophiliccyanobacterium Synechococcus elongatus [see, Coufal J, Hladik J andSofrova D (1989)]. The carotenoid content of photosystem 1pigment-protein complexes of the cyanobacterium Synechococcus elongatus.Photosynthetica 23: 603-616]. A subunit protein-complex structure of PSI from the thermophilic cyanobacterium Synechococcus sp., whichconsisted of four polypeptides (of 62, 60, 14 and 10 kDa), containedapproximately 10 β-carotene molecules per P700 [see, Takahashi Y, HirotaK and Katoh S (1985) Multiple forms of P700-chlorophyll α-proteincomplexes from Synechococcus sp.: the iron, quinone and carotenoidcontents. Photosynth Res 6: 183-192]. This carotenoid is exclusivelybound to the large polypeptides which carry the functional and antennachlorophyll a. The fluorescence excitation spectrum of these complexessuggested that α-carotene serves as an efficient antenna for PS I.

As mentioned, an additional essential function of carotenoids is toprotect against photooxidation processes in the photosynthetic apparatusthat are caused by the excited triplet state of chlorophyll. Carotenoidmolecules with π-electron conjugation of nine or more carbon-carbondouble bonds can absorb triplet-state energy from chlorophyll and thusprevent the formation of harmful singlet-state oxygen radicals. InSynechococcus sp. the triplet state of carotenoids was monitored inclosed PS II centers and its rise kinetics of approximately 25nanoseconds is attributed to energy transfer from chlorophyll tripletsin the antenna [see, Schlodder E and Brettel K (1988) Primary chargeseparation in closed photosystem II with a lifetime of 11 nanoseconds.Flash-absorption spectroscopy with oxygen-evolving photosystem IIcomplexes from Synechococcus. Biochim Biophys Acta 933: 22-34]. It isconceivable that this process, that has a lower yield compared to theyield of radical-pair formation, plays a role in protecting chlorophyllfrom damage due to over-excitation.

The protective role of carotenoids in vivo has been elucidated throughthe use of bleaching herbicides such as norflurazon that inhibitcarotenoid biosynthesis in all organisms performing oxygenicphotosynthesis [reviewed by Sandmann G and Boger P (1989) Inhibition ofcarotenoid biosynthesis by herbicides. In: Boger P and Sandmann G (Eds.)Target Sites of Herbicide Action, pp 25-44. CRC Press, Boca Raton,Fla.]. Treatment with norflurazon in the light results in a decrease ofboth carotenoid and chlorophyll levels, while in the dark, chlorophylllevels are unaffected. Inhibition of photosynthetic efficiency in cellsof Oscillatoria agardhii that were treated with the pyridinoneherbicide, fluridone, was attributed to a decrease in the relativeabundance of myxoxanthophyll, zeaxanthin and β-carotene, which in turncaused photooxidation of chlorophyll molecules [see, Canto de Loura I,Dubacq J P and Thomas J C (1987) The effects of nitrogen deficiency onpigments and lipids of cianobacteria. Plant Physiol 83: 838-843].

It has been demonstrated in plants that zeaxanthin is required todissipate, in a nonradiative manner, the excess excitation energy of theantenna chlorophyll [see, Demmig-Adams B (1990) Carotenoids andphotoprotection in plants: a role for the xanthophyll zeaxanthin.Biochim Biophys Acta 1020: 1-24; and, Demmig-Adams B and Adams W W III(1990) The carotenoid zeaxanthin and high-energy-state quenching ofchlorophyll fluorescence. Photosynth Res 25: 187-197]. In algae andplants a light-induced deepoxidation of violaxanthin to yieldzeaxanthin, is related to photoprotection processes [reviewed byDemmig-Adams B and Adams W W III (1992) Photoprotection and otherresponses of plants to high light stress. Ann Rev Plant Physiol PlantMol Biol 43: 599-626]. The light-induced deepoxidation of violaxanthinand the reverse reaction that takes place in the dark, are known as the“xanthophyll cycle” [see, Demmig-Adams B and Adams W W III (1992)Photoprotection and other responses of plants to high light stress. AnnRev Plant Physiol Plant Mol Biol 43: 599-626]. Cyanobacterial lichens,that do not contain any zeaxanthin and that probably are incapable ofradiation energy dissipation, are sensitive to high light intensity;algal lichens that contain zeaxanthin are more resistant to high-lightstress [see, Demmig-Adams B, Adams W W III, Green T G A, Czygan F C andLange O L (1990) Differences in the susceptibility to light stress intwo lichens forming a phycosymbiodeme, one partner possessing and onelacking the xanthophyll cycle. Oecologia 84: 451-456; Demmig-Adams B andAdams W W III (1993) The xanthophyll cycle, protein turnover, and thehigh light tolerance of sun-acclimated leaves. Plant Physiol 103:1413-1420; and, Demmig-Adams B (1990) Carotenoids and photoprotection inplants: a role for the xanthophyll zeaxanthin. Biochim Biophys Acta1020: 1-24]. In contrast to algae and plants, cyanobacteria do not havea xanthophyll cycle. However, they do contain ample quantities ofzeaxanthin and other xanthophylls that can support photoprotection ofchlorophyll.

Several other functions have been ascribed to carotenoids. Thepossibility that carotenoids protect against damaging species generatedby near ultra-violet (UV) irradiation is suggested by results describingthe accumulation of β-carotene in a UV-resistant mutant of thecyanobacterium Gloeocapsa alpicola [see, Buckley C E and Houghton J A(1976) A study of the effects of near UV radiation on the pigmentationof the blue-green alga Gloeocapsa alpicola. Arch Microbiol 107: 93-97].This has been demonstrated more elegantly in Escherichia coli cells thatproduce carotenoids [see, Tuveson R W and Sandmann G (1993) Protectionby cloned carotenoid genes expressed in Escherichia coli againstphototoxic molecules activated by near-ultraviolet light. Meth Enzymol214: 323-330]. Due to their ability to quench oxygen radical species,carotenoids are efficient anti-oxidants and thereby protect cells fromoxidative damage. This function of carotenoids is important in virtuallyall organisms [see, Krinsky N I (1989) Antioxidant functions ofcarotenoids. Free Radical Biol Med 7: 617-635; and, Palozza P andKrinsky N I (1992) Antioxidant effects of carotenoids in vivo and invitro—an overview. Meth Enzymol 213: 403-420]. Other cellular functionscould be affected by carotenoids, even if indirectly.

In flowers and fruits carotenoids facilitate the attraction ofpollinators and dispersal of seeds. This latter aspect is stronglyassociated with agriculture. The type and degree of pigmentation infruits and flowers are among the most important traits of many crops.This is mainly since the colors of these products often determine theirappeal to the consumers and thus can increase their market worth.

Carotenoids have important commercial uses as coloring agents in thefood industry since they are non-toxic [see, Bauernfeind J C (1981)Carotenoids as colorants and vitamin A precursors. Academic Press,London]. The red color of the tomato fruit is provided by lycopene whichaccumulates during fruit ripening in chromoplasts. Tomato extracts,which contain high content (over 80% dry weight) of lycopene, arecommercially produced worldwide for industrial use as food colorant.Furthermore, the flesh, feathers or eggs of fish and birds assume thecolor of the dietary carotenoid provided, and thus carotenoids arefrequently used in dietary additives for poultry and in aquaculture.Certain cyanobacterial species, for example Spirulina sp. [see, Sommer TR, Potts W T and Morrissy N M (1990) Recent progress in processedmicroalgae in aquaculture. Hydrobiologia 204/205: 435-443], arecultivated in aquaculture for the production of animal and human foodsupplements. Consequently, the content of carotenoids, primarily ofβ-carotene, in these cyanobacteria has a major commercial implication inbiotechnology.

Most carotenoids are composed of a C₄₀ hydrocarbon backbone, constructedfrom eight C₅ isoprenoid units and contain a series of conjugated doublebonds. Carotenes do not contain oxygen atoms and are either linear orcyclized molecules containing one or two end rings. Xanthophylls areoxygenated derivatives of carotenes. Various glycosilated carotenoidsand carotenoid esters have been identified. The C₄₀ backbone can befurther extended to give C₄₅ or C₅₀ carotenoids, or shortened yieldingapocarotenoids. Some nonphotosynthetic bacteria also synthesize C₃₀carotenoids. General background on carotenoids can be found in Goodwin TW (1980) The Biochemistry of the Carotenoids, Vol. 1, 2nd Ed. Chapmanand Hall, New York; and in Goodwin T W and Britton G (1988) Distributionand analysis of carotenoids. In: Goodwin T W (ed) Plant Pigments, pp62-132. Academic Press, New York.

More than 640 different naturally-occurring carotenoids have been so farcharacterized, hence, carotenoids are responsible for most of thevarious shades of yellow, orange and red found in microorganisms, fungi,algae, plants and animals. Carotenoids are synthesized by allphotosynthetic organisms as well as several nonphotosynthetic bacteriaand fungi, however they are also widely distributed through feedingthroughout the animal kingdom.

Carotenoids are synthesized de novo from isoprenoid precursors only inphotosynthetic organisms and some microorganisms, they typicallyaccumulate in protein complexes in the photosynthetic membrane, in thecell membrane and in the cell wall.

In the biosynthesis pathway of β-carotene, four enzymes convertgeranylgeranyl pyrophosphate of the central isoprenoid pathway toβ-carotene. Carotenoids are produced from the general isoprenoidbiosynthetic pathway. While this pathway has been known for severaldecades, only recently, and mainly through the use of genetics andmolecular biology, have some of the molecular mechanisms involved incarotenoids biogenesis, been elucidated. This is due to the fact thatmost of the enzymes which take part in the conversion of phytoene tocarotenes and xanthophylls are labile, membrane-associated proteins thatlose activity upon solubilization [see, Beyer P, Weiss G and Kleinig H(1985) Solubilization and reconstitution of the membrane-boundcarotenogenic enzymes from daffodile chromoplasts. Eur J Biochem 153:341-346; and, Bramley P M (1985) The in vitro biosynthesis ofcarotenoids. Adv Lipid Res 21: 243-279].

Carotenoids are synthesized from isoprenoid precursors. The centralpathway of isoprenoid biosynthesis may be viewed as beginning with theconversion of acetyl-CoA to mevalonic acid. D³-isopentenyl pyrophosphate(IPP), a C₅ molecule, is formed from mevalonate and is the buildingblock for all long-chain isoprenoids. Following isomerization of IPP todimethylallyl pyrophosphate (DMAPP), three additional molecules of IPPare combined to yield the C20 molecule, geranylgeranyl pyrophosphate(GGPP). These 1′-4 condensation reactions are catalyzed by prenyltransferases [see, Kleinig H (1989) The role of plastids in isoprenoidbiosynthesis. Ann Rev Plant Physiol Plant Mol Biol 40: 39-59]. There isevidence in plants that the same enzyme, GGPP synthase, carries out allthe reactions from DMAPP to GGPP [see, Dogbo O and Camara B (1987)Purification of isopentenyl pyrophosphate isomerase and geranylgeranylpyrophosphate synthase from Capsicum chromoplasts by affinitychromatography. Biochim Biophys Acta 920: 140-148; and, Laferriere A andBeyer P (1991) Purification of geranylgeranyl diphosphate synthase fromSinapis alba etioplasts. Biochim Biophys Acta 216: 156-163].

The first step that is specific for carotenoid biosynthesis is thehead-to-head condensation of two molecules of GGPP to produceprephytoene pyrophosphate (PPPP). Following removal of thepyrophosphate, GGPP is converted to 15-cis-phytoene, a colorless C₄₀hydrocarbon molecule. This two-step reaction is catalyzed by the solubleenzyme, phytoene synthase, an enzyme encoded by a single gene (crtB), inboth cyanobacteria and plants [see, Chamovitz D, Misawa N, Sandmann Gand Hirschberg J (1992) Molecular cloning and expression in Escherichiacoli of a cyanobacterial gene coding for phytoene synthase, a carotenoidbiosynthesis enzyme. FEBS Lett 296: 305-310; Ray J A, Bird C R, MaundersM, Grierson D and Schuch W (1987) Sequence of pTOM5, a ripening relatedcDNA from tomato. Nucl Acids Res 15: 10587-10588; Camara B (1993) Plantphytoene synthase complex—component 3 enzymes, immunology, andbiogenesis. Meth Enzymol 214: 352-365]. All the subsequent steps in thepathway occur in membranes. Four desaturation (dehydrogenation)reactions convert phytoene to lycopene via phytofluene, ζ-carotene, andneurosporene. Each desaturation increases the number of conjugateddouble bonds by two such that the number of conjugated double bondsincreases from three in phytoene to eleven in lycopene.

Relatively little is known about the molecular mechanism of theenzymatic dehydrogenation of phytoene [see, Jones B L and Porter J W(1986) Biosynthesis of carotenes in higher plants. CRC Crit Rev PlantSci 3: 295-324; and, Beyer P, Mayer M and Kleinig H (1989) Molecularoxygen and the state of geometric iosomerism of intermediates areessential in the carotene desaturation and cyclization reactions indaffodil chromoplasts. Eur J Biochem 184: 141-150]. It has beenestablished that in cyanobacteria, algae and plants the first twodesaturations, from 15-cis-phytoene to ζ-carotene, are catalyzed by asingle membrane-bound enzyme, phytoene desaturase [see, Jones B L andPorter J W (1986) Biosynthesis of carotenes in higher plants. CRC CritRev Plant Sci 3: 295-324; and, Beyer P, Mayer M and Kleinig H (1989)Molecular oxygen and the state of geometric iosomerism of intermediatesare essential in the carotene desaturation and cyclization reactions indaffodil chromoplasts. Eur J Biochem 184: 141-150]. Since the 1-caroteneproduct is mostly in the all-trans configuration, a cis-transisomerization is presumed at this desaturation step. The primarystructure of the phytoene desaturase polypeptide in cyanobacteria isconserved (over 65% identical residues) with that of algae and plants[see, Pecker I, Chamovitz D, Linden H, Sandmann G and Hirschberg J(1992) A single polypeptide catalyzing the conversion of phytoene toζ-carotene is transcriptionally regulated during tomato fruit ripening.Proc Natl Acad Sci USA 89: 4962-4966; Pecker I, Chamovitz D, Mann V,Sandmann G, Boger P and Hirschberg J (1993) Molecular characterizationof carotenoid biosynthesis in plants: the phytoene desaturase gene intomato. In: Murata N (ed) Research in Photosynthesis, Vol III, pp 11-18.Kluwer, Dordrectht]. Moreover, the same inhibitors block phytoenedesaturase in the two systems [see, Sandmann G and Boger P (1989)Inhibition of carotenoid biosynthesis by herbicides. In: Boger P andSandmann G (eds) Target Sites of Herbicide Action, pp 25-44. CRC Press,Boca Raton, Fla.]. Consequently, it is very likely that the enzymescatalyzing the desaturation of phytoene and phytofluene in cyanobacteriaand plants have similar biochemical and molecular properties, that aredistinct from those of phytoene desaturases in other microorganisms. Onesuch a difference is that phytoene desaturases from Rhodobactercapsulatus, Erwinia sp. or fungi convert phytoene to neurosporene,lycopene, or 3,4-dehydrolycopene, respectively.

Desaturation of phytoene in daffodil chromoplasts [see, Beyer P, Mayer Mand Keinig H (1989) Molecular oxygen and the state of geometriciosomerism of intermediates are essential in the carotene desaturationand cyclization reactions in daffodil chromoplasts. Eur J Biochem 184:141-150], as well as in a cell free system of Synechococcus sp. strainPCC 7942 [see, Sandmann G and Kowalczyk S (1989) In vitrocarotenogenesis and characterization of the phytoene desaturase reactionin Anacystis. Biochem Biophys Res Corn 163: 916-921], is dependent onmolecular oxygen as a possible final electron acceptor, although oxygenis not directly involved in this reaction. A mechanism ofdehydrogenase-electron transferase was supported in cyanobacteria overdehydrogenation mechanism of dehydrogenase-monooxygenase [see, SandmannG and Kowalczyk S (1989) In vitro carotenogenesis and characterizationof the phytoene desaturase reaction in Anacystis. Biochem Biophys ResCom 163: 916-921]. A conserved FAD-binding motif exists in all phytoenedesaturases whose primary structures have been analyzed [see, Pecker I,Chamovitz D, Linden H, Sandmann G and Hirschberg J (1992) A singlepolypeptide catalyzing the conversion of phytoene to ζ-carotene istranscriptionally regulated during tomato fruit ripening. Proc Natl AcadSci USA 89: 4962-4966; Pecker I, Chamovitz D, Mann V, Sandmann G, BogerP and Hirschberg J (1993) Molecular characterization of carotenoidbiosynthesis in plants: the phytoene desaturase gene in tomato. In:Murata N (ed) Research in Photosynthesis, Vol III, pp 11-18. Kluwer,Dordrectht]. The phytoene desaturase enzyme in pepper was shown tocontain a protein-bound FAD [see, Hugueney P, Romer S, Kuntz M andCamara B (1992) Characterization and molecular cloning of a flavoproteincatalyzing the synthesis of phytofluene and ζ-carotenein Capsicumchromoplasts. Eur J Biochem 209: 399-407]. Since phytoene desaturase islocated in the membrane, an additional, soluble redox component ispredicted. This hypothetical component could employ NAD(P)⁺, assuggested [see, Mayer M P, Nievelstein V and Beyer P (1992) Purificationand characterization of a NADPH dependent oxidoreductase fromchromoplasts of Narcissus pseudonarcissus—a redox-mediator possiblyinvolved in carotene desaturation. Plant Physiol Biochem 30: 389-398] oranother electron and hydrogen carrier, such as a quinone. The cellularlocation of phytoene desaturase in Synechocystis sp. strain PCC 6714 andAnabaena variabilis strain ATCC 29413 was determined with specificantibodies to be mainly (85%) in the photosynthetic thylakoid membranes[see, Serrano A, Gimenez P, Schmidt A and Sandmann G (1990)Immunocytochemical localization and functional determination of phytoenedesaturase in photoautotrophic prokaryotes. J Gen Microbiol 136:2465-2469].

In cyanobacteria algae and plants ζ-carotene is converted to lycopenevia neurosporene. Very little is known about the enzymatic mechanism,which is predicted to be carried out by a single enzyme [see, Linden H,Vioque A and Sandmann G (1993) Isolation of a carotenoid biosynthesisgene coding for ζ-carotene desaturase from Anabaena PCC 7120 byheterologous complementation. FEMS Microbiol Lett 106: 99-104]. Thededuced amino acid sequence of ζ-carotene desaturase in Anabaena sp.strain PCC 7120 contains a dinucleotide-binding motif that is similar tothe one found in phytoene desaturase.

Two cyclization reactions convert lycopene to β-carotene. Evidence hasbeen obtained that in Synechococcus sp. strain PCC 7942 [see, CunninghamF X Jr, Chamovitz D, Misawa N, Gantt E and Hirschberg J (1993) Cloningand functional expression in Escherichia coli of a cyanobacterial genefor lycopene cyclase, the enzyme that catalyzes the biosynthesis ofβ-carotene. FEBS Lett 328: 130-138], as well as in plants [see, Camara Band Dogbo O (1986) Demonstration and solubilization of lycopene cyclasefrom Capsicum chromoplast membranes. Plant Physiol 80: 172-184], thesetwo cyclizations are catalyzed by a single enzyme, lycopene cyclase.This membrane-bound enzyme is inhibited by the triethylamine compounds,CPTA and MPTA [see, Sandmann G and Boger P (1989) Inhibition ofcarotenoid biosynthesis by herbicides. In: Boger P and Sandmann G (eds)Target Sites of Herbicide Action, pp 25-44. CRC Press, Boca Raton,Fla.]. Cyanobacteria carry out only the α-cyclization and therefore donot contain 1-carotene, 6-carotene and α-carotene and their oxygenatedderivatives. The β-ring is formed through the formation of a “carboniumion” intermediate when the C-1,2 double bond at the end of the linearlycopene molecule is folded into the position of the C-5,6 double bond,followed by a loss of a proton from C-6. No cyclic carotene has beenreported in which the 7,8 bond is not a double bond. Therefore, fulldesaturation as in lycopene, or desaturation of at least half-moleculeas in neurosporene, is essential for the reaction. Cyclization oflycopene involves a dehydrogenation reaction that does not requireoxygen. The cofactor for this reaction is unknown. Adinucleotide-binding domain was found in the lycopene cyclasepolypeptide of Synechococcus sp. strain PCC 7942, implicating NAD(P) orFAD as coenzymes with lycopene cyclase.

The addition of various oxygen-containing side groups, such as hydroxy-,methoxy-, oxo-, epoxy-, aldehyde or carboxylic acid moieties, form thevarious xanthophyll species. Little is known about the formation ofxanthophylls. Hydroxylation of β-carotene requires molecular oxygen in amixed-function oxidase reaction.

Clusters of genes encoding the enzymes for the entire pathway have beencloned from the purple photosynthetic bacterium Rhodobacter capsulatus[see, Armstrong G A, Alberti M, Leach F and Hearst J E (1989) Nucleotidesequence, organization, and nature of the protein products of thecarotenoid biosynthesis gene cluster of Rhodobacter capsulatus. Mol GenGenet 216: 254-268] and from the nonphotosynthetic bacteria Erwiniaherbicola [see, Sandmann G, Woods W S and Tuveson R W (1990)Identification of carotenoids in Erwinia herbicola and in transformedEscherichia coli strain. FEMS Microbiol Lett 71: 77-82; Hundle B S,Beyer P, Kleinig H, Englert H and Hearst J E (1991) Carotenoids ofErwinia herbicola and an Escherichia coli HB11 strain carrying theErwinia herbicola carotenoid gene cluster. Photochem Photobiol 54:89-93; and, Schnurr G, Schmidt A and Sandmann G (1991) Mapping of acarotenogenic gene cluster from Erwinia herbicola and functionalidentification of six genes. FEMS Microbiol Lett 78: 157-162] andErwinia uredovora [see, Misawa N, Nakagawa M, Kobayashi K, Yamano S,Izawa I, Nakamura K and Harashima K (1990) Elucidation of the Erwiniauredovora carotenoid biosynthetic pathway by functional analysis of geneproducts in Escherichia coli. J Bacteriol 172: 6704-6712]. Two genes,al-3 for GGPP synthase [see, Nelson M A, Morelli G, Carattoli A, RomanoN and Macino G (1989) Molecular cloning of a Neurospora crassacarotenoid biosynthetic gene (albino-3) regulated by blue light and theproducts of the white collar genes. Mol Cell Biol 9: 1271-1276; and,Carattoli A, Romano N, Ballario P, Morelli G and Macino G (1991) TheNeurospora crassa carotenoid biosynthetic gene (albino 3). J Biol Chem266: 5854-5859] and al-1 for phytoene desaturase [see, Schmidhauser T J,Lauter F R, Russo V EA and Yanofsky C (1990) Cloning sequencing andphotoregulation of al-1, a carotenoid biosynthetic gene of Neurosporacrassa. Mol Cell Biol 10: 5064-5070] have been cloned from the fungusNeurospora crassa. However, attempts at using these genes asheterologous molecular probes to clone the corresponding genes fromcyanobacteria or plants were unsuccessful due to lack of sufficientsequence similarity.

The first “plant-type” genes for carotenoid synthesis enzyme were clonedfrom cyanobacteria using a molecular-genetics approach. In the firststep towards cloning the gene for phytoene desaturase, a number ofmutants that are resistant to the phytoene-desaturase-specificinhibitor, norflurazon, were isolated in Synechococcus sp. strain PCC7942 [see, Linden H, Sandmann G, Chamovitz D, Hirschberg J and Boger P(1990) Biochemical characterization of Synechococcus mutants selectedagainst the bleaching herbicide norflurazon. Pestic Biochem Physiol 36:46-51]. The gene conferring norflurazon-resistance was then cloned bytransforming the wild-type strain to herbicide resistance [see,Chamovitz D, Pecker I and Hirschberg J (1991) The molecular basis ofresistance to the herbicide norflurazon. Plant Mol Biol 16: 967-974;Chamovitz D, Pecker I, Sandmann G, Boger P and Hirschberg J (1990)Cloning a gene for norflurazon resistance in cyanobacteria. ZNaturforsch 45c: 482-486]. Several lines of evidence indicated that thecloned gene, formerly called pds and now named crtP, codes for phytoenedesaturase. The most definitive one was the functional expression ofphytoene desaturase activity in transformed Escherichia coli cells [see,Linden H, Misawa N, Chamovitz D, Pecker I, Hirschberg J and Sandmann G(1991) Functional complementation in Escherichia coli of differentphytoene desaturase genes and analysis of accumulated carotenes. ZNaturforsch 46c: 1045-1051; and, Pecker I, Chamovitz D, Linden H,Sandmann G and Hirschberg J (1992) A single polypeptide catalyzing theconversion of phytoene to ζ-carotene is transcriptionally regulatedduring tomato fruit ripening. Proc Natl Acad Sci USA 89: 4962-4966]. ThecrtP gene was also cloned from Synechocystis sp. strain PCC 6803 bysimilar methods [see, Martinez-Ferez I M and Vioque A (1992) Nucleotidesequence of the phytoene desaturase gene from Synechocystis sp. PCC 6803and characterization of a new mutation which confers resistance to theherbicide norflurazon. Plant Mol Biol 18: 981-983].

The cyanobacterial crtP gene was subsequently used as a molecular probefor cloning the homologous gene from an alga [see, Pecker I, ChamovitzD, Mann V, Sandmann G, Boger P and Hirschberg J (1993) Molecularcharacterization of carotenoid biosynthesis in plants: the phytoenedesaturase gene in tomato. In: Murata N (ed) Research in Photosynthesis,Vol III, pp 11-18. Kluwer, Dordrectht] and higher plants [see, Bartley GE, Viitanen P V, Pecker I, Chamovitz D, Hirschberg J and Scolnik P A(1991) Molecular cloning and expression in photosynthetic bacteria of asoybean cDNA coding for phytoene desaturase, an enzyme of the carotenoidbiosynthesis pathway. Proc Natl Acad Sci USA 88: 6532-6536; and, PeckerI, Chamovitz D, Linden H, Sandmann G and Hirschberg J (1992) A singlepolypeptide catalyzing the conversion of phytoene to ζ-carotene istranscriptionally regulated during tomato fruit ripening. Proc Natl AcadSci USA 89: 4962-4966]. The phytoene desaturases in Synechococcus sp.strain PCC 7942 and Synechocystis sp. strain PCC 6803 consist of 474 and467 amino acid residues, respectively, whose sequences are highlyconserved (74% identities and 86% similarities). The calculatedmolecular mass is 51 kDa and, although it is slightly hydrophobic(hydropathy index −0.2), it does not include a hydrophobic region whichis long enough to span a lipid bilayer membrane. The primary structureof the cyanobacterial phytoene desaturase is highly conserved with theenzyme from the green alga Dunalliela bardawil (61% identical and 81%similar; [see, Pecker I, Chamovitz D, Mann V, Sandmann G, Boger P andHirschberg J (1993) Molecular characterization of carotenoidbiosynthesis in plants: the phytoene desaturase gene in tomato. In:Murata N (ed) Research in Photosynthesis, Vol III, pp 11-18. Kluwer,Dordrectht]) and from tomato [see, Pecker I, Chamovitz D, Linden H,Sandmann G and Hirschberg J (1992) A single polypeptide catalyzing theconversion of phytoene to ζ-carotene is transcriptionally regulatedduring tomato fruit ripening. Proc Natl Acad Sci USA 89: 4962-4966],pepper [see, Hugueney P, Romer S, Kuntz M and Camara B (1992)Characterization and molecular cloning of a flavoprotein catalyzing thesynthesis of phytofluene and ζ-carotenein Capsicum chromoplasts. Eur JBiochem 209: 399-407] and soybean [see, Bartley G E, Viitanen P V,Pecker I, Chamovitz D, Hirschberg J and Scolnik P A (1991) Molecularcloning and expression in photosynthetic bacteria of a soybean cDNAcoding for phytoene desaturase, an enzyme of the carotenoid biosynthesispathway. Proc Natl Acad Sci USA 88: 6532-6536] (62-65% identical and˜79% similar; [see, Chamovitz D (1993) Molecular analysis of the earlysteps of carotenoid biosynthesis in cyanobacteria: Phytoene synthase andphytoene desaturase. Ph.D. Thesis, The Hebrew University of Jerusalem]).The eukaryotic phytoene desaturase polypeptides are larger (64 kDa);however, they are processed during import into the plastids to matureforms whose sizes are comparable to those of the cyanobacterial enzymes.

There is a high degree of structural similarity in carotenoid enzymes ofRhodobacter capsulatus, Erwinia sp. and Neurospora crassa [reviewed inArmstrong G A, Hundle B S and Hearst J E (1993) Evolutionaryconservation and structural similarities of carotenoid biosynthesis geneproducts from photosynthetic and nonphotosynthetic organisms. MethEnzymol 214: 297-311], including in the crtI gene-product, phytoenedesaturase. As indicated above, a high degree of conservation of theprimary structure of phytoene desaturases also exists among oxygenicphotosynthetic organisms. However, there is little sequence similarity,except for the FAD binding sequences at the amino termini, between the“plant-type” crtP gene products and the “bacterial-type” phytoenedesaturases (crtI gene products; 19-23% identities and 42-47%similarities). It has been hypothesized that crtP and crtI are notderived from the same ancestral gene and that they originatedindependently through convergent evolution [see, Pecker I, Chamovitz D,Linden H, Sandmann G and Hirschberg J (1992) A single polypeptidecatalyzing the conversion of phytoene to ζ-carotene is transcriptionallyregulated during tomato fruit ripening. Proc Natl Acad Sci USA 89:4962-4966]. This hypothesis is supported by the differentdehydrogenation sequences that are catalyzed by the two types of enzymesand by their different sensitivities to inhibitors.

Although not as definite as in the case of phytoene desaturase, asimilar distinction between cyanobacteria and plants on the one hand andother microorganisms is also seen in the structure of phytoene synthase.The crtB gene (formerly psy) encoding phytoene synthase was identifiedin the genome of Synechococcus sp. strain PCC 7942 adjacent to crtP andwithin the same operon [see, Bartley G E, Viitanen P V, Pecker I,Chamovitz D, Hirschberg J and Scolnik P A (1991) Molecular cloning andexpression in photosynthetic bacteria of a soybean cDNA coding forphytoene desaturase, an enzyme of the carotenoid biosynthesis pathway.Proc Natl Acad Sci USA 88: 6532-6536]. This gene encodes a 36-kDapolypeptide of 307 amino acids with a hydrophobic index of −0.4. Thededuced amino acid sequence of the cyanobacterial phytoene synthase ishighly conserved with the tomato phytoene synthase (57% identical and70% similar; Ray J A, Bird C R, Maunders M, Grierson D and Schuch W(1987) Sequence of pTOM5, a ripening related cDNA from tomato. NuclAcids Res 15: 10587-10588]) but is less highly conserved with the crtBsequences from other bacteria (29-32% identical and 48-50% similar withten gaps in the alignment). Both types of enzymes contain two conservedsequence motifs also found in prenyl transferases from diverse organisms[see, Bartley G E, Viitanen P V, Pecker I, Chamovitz D, Hirschberg J andScolnik P A (1991) Molecular cloning and expression in photosyntheticbacteria of a soybean cDNA coding for phytoene desaturase, an enzyme ofthe carotenoid biosynthesis pathway. Proc Natl Acad Sci USA 88:6532-6536; Carattoli A, Romano N, Ballario P, Morelli G and Macino G(1991) The Neurospora crassa carotenoid biosynthetic gene (albino 3). JBiol Chem 266: 5854-5859; Armstrong G A, Hundle B S and Hearst J E(1993) Evolutionary conservation and structural similarities ofcarotenoid biosynthesis gene products from photosynthetic andnonphotosynthetic organisms. Meth Enzymol 214: 297-311; Math S K, HearstJ E and Poulter C D (1992) The crtE gene in Erwinia herbicola encodesgeranylgeranyl diphosphate synthase. Proc Natl Acad Sci USA 89:6761-6764; and, Chamovitz D (1993) Molecular analysis of the early stepsof carotenoid biosynthesis in cyanobacteria: Phytoene synthase andphytoene desaturase. Ph.D. Thesis, The Hebrew University of Jerusalem].It is conceivable that these regions in the polypeptide are involved inthe binding and/or removal of the pyrophosphate during the condensationof two GGPP molecules.

The crtQ gene encoding 1-carotene desaturase (formerly zds) was clonedfrom Anabaena sp. strain PCC 7120 by screening an expression library ofcyanobacterial genomic DNA in cells of Escherichia coli carrying theErwinia sp. crtB and crtE genes and the cyanobacterial crtP gene [see,Linden H, Vioque A and Sandmann G (1993) Isolation of a carotenoidbiosynthesis gene coding for ζ-carotene desaturase from Anabaena PCC7120 by heterologous complementation. FEMS Microbiol Lett 106: 99-104].Since these Escherichia coli cells produce ζ-carotene, brownish-redpigmented colonies that produced lycopene could be identified on theyellowish background of cells producing ζ-carotene. The predictedζ-carotene desaturase from Anabaena sp. strain PCC 7120 is a 56-kDapolypeptide which consists of 499 amino acid residues. Surprisingly, itsprimary structure is not conserved with the “plant-type” (crtP geneproduct) phytoene desaturases, but it has considerable sequencesimilarity to the bacterial-type enzyme (crtI gene product) [see,Sandmann G (1993) Genes and enzymes involved in the desaturationreactions from phytoene to lycopene. (abstract), 10th InternationalSymposium on Carotenoids, Trondheim CL1-2]. It is possible that thecyanobacterial crtQ gene and crtI gene of other microorganismsoriginated in evolution from a common ancestor.

The crtL gene for lycopene cyclase (formerly lcy) was cloned fromSynechococcus sp. strain PCC 7942 utilizing essentially the same cloningstrategy as for crtP. By using an inhibitor of lycopene cyclase,2-(4-methylphenoxy)-triethylamine hydrochloride (MPTA), the gene wasisolated by transformation of the wild-type to herbicide-resistance[see, Cunningham F X Jr, Chamovitz D, Misawa N, Gantt E and Hirschberg J(1993) Cloning and functional expression in Escherichia coli of acyanobacterial gene for lycopene cyclase, the enzyme that catalyzes thebiosynthesis of β-carotene. FEBS Lett 328: 130-138]. Lycopene cyclase isthe product of a single gene product and catalyzes the doublecyclization reaction of lycopene to α-carotene. The crtL gene product inSynechococcus sp. strain PCC 7942 is a 46-kDa polypeptide of 411 aminoacid residues. It has no sequence similarity to the crtY gene product(lycopene cyclase) from Erwinia uredovora or Erwinia herbicola.

The gene for β-carotene hydroxylase (crtZ) and zeaxanthin glycosilase(crtX) have been cloned from Erwinia herbicola [see, Hundle B, AlbertiM, Nievelstein V, Beyer P, Kleinig H, Armstrong G A, Burke D H andHearst J E (1994) Functional assignment of Erwinia herbicola Eho10carotenoid genes expressed in Escherichia coli. Mol Gen Genet 254:406-416; Hundle B S, Obrien D A, Alberti M, Beyer P and Hearst J E(1992) Functional expression of zeaxanthin glucosyltransferase fromErwinia herbicola and a proposed diphosphate binding site. Proc NatlAcad Sci USA 89: 9321-9325] and from Erwinia uredovora [see, Misawa N,Nakagawa M, Kobayashi K, Yamano S, Izawa I, Nakamura K and Harashima K(1990) Elucidation of the Erwinia uredovora carotenoid biosyntheticpathway by functional analysis of gene products in Escherichia coli. JBacteriol 172: 6704-6712].

Carotenoids as Antioxidants:

Most carotenoids are efficient antioxidants, quenching singlet oxygen(102) and scavenging peroxyl radicals (Sies and Stahl, 1995). ¹O₂, O₂ ⁻,H₂O₂ and peroxyl radicals are reactive oxygen species generated inbiological cells. All these species may react with DNA, proteins andlipids impairing their physiological functions (Halliwell, 1996). Suchprocesses are discussed as initial events in the pathogenesis of severaldiseases including cancer, cardiovascular diseases, or age-relatedsystem degeneration. Carotenoids inactivate singlet oxygen via physicalor chemical quenching. The efficacy of physical quenching exceeds thatof chemical quenching by far, 99.9%, and involves that transfer ofexcitation energy from ¹O₂ to the carotenoid. In the process of physicalquenching the carotenoid remains intact, so that it can undergo furthercycles of singlet oxygen quenching. Methylene blue was used as asensitizer to study the consumption of carotenoids during photooxidationof human plasma and LDL (Ojima et al., 1993). Lycopene, β-carotene andxanthophylls were found to decrease photooxidation in blood plasma whilethey remained unchanged (Wagner et al., 1993). Hirayama et al. (1994)investigated the singlet oxygen quenching ability of 18 carotenoids andreported that the xanthophylls conjugated keto group enhanced quenching,while hydroxy, epoxy and methoxy groups showed lesser effects.

Capsanthin and capsorubin were found to act as better singlet oxygenquenchers than β-carotene. Previous studies show that β-carotene is agood scavenger of hypochlorite and others have demonstrated itsscavenging ability of nitrogen dioxide. (Kanner et al., 1983, Everett etal., 1996).

Carotenoids are efficient scavengers of peroxyl radicals, especially atlow oxygen tension (Burton and Ingold, 1984; Kennedy and Liebler, 1992).The interaction of carotenoids with peroxyl radicals generated by theazo compounds AMVN and AAPH in a phosphatidylcholine liposome systemwere investigated by Lin et al. (1992). In this system the xanthophyllsastaxanthin, zeaxanthin and cantaxanthin were more efficient freeradical scavengers than β-carotene. However, investigating the reactionof carotenoids with peroxyl free radical in emulsion showed thatlycopene and β-carotene are better scavengers than several xanthophylls(Woodall et al., 1997). Matsufuji et al. (1998) investigated the radicalscavenging ability of carotenoids in methyl linoleate emulsion anddemonstrated that capsanthin acts better than lutein, zeaxanthin andβ-carotene.

Oxidative modification of low-density lipoproteins (LDL), which isthought to be a key step in early atherosclerosis, is protected by thelipoprotein-associated antioxidants. LDL contains about 1 carotenoid and12 α-tocopherol molecules per LDL particle, a relatively small numbercompared with about 2,300 molecules of oxidizable lipid in each LDLparticle (Romanchik et al., 1995). Some antioxidant supplements, such asα-tocopherol consistently appear to enhance the ability of LDL to resistoxidation, (Esterbauer et al., 1991; Aviram, 1999). However, β-caroteneshows less consistent protective ability (Gaziano et al., 1995; Reavenet al., 1994). In contrast, Lin et al. (1998) showed that depletion ofβ-carotene in healthy women increased the susceptibility of LDL tooxidation, whereas a normal intake provide protection to LDL. Mostrecently, dietary supplementation with β-carotene, but not lycopene wasshown to inhibit endothelial cell—mediated ex-vivo per oxidation of LDL(Dugas et al., 1999). Mixture of carotenoids have been found to be moreeffective than any single carotenoid in protecting liposomes againstlipid peroxidation (Stahl et al., 1998), and as antioxidants inmembranes and LDL. Moreover, it has been reported that carotenoidsenhance vitamin E antioxidant efficiency (Bohm et al., 1997; Fuhrman etal., 1997; Fuhrman and Aviram, 1999).

Atherosclerosis and LDL Oxidation as Affected by Carotenoids duringAtherogenesis:

Atherosclerosis is the major cause of morbidity and mortality in thewestern world and its pathogenesis involves complicated interactingamong cells of the arterial wall, blood cells, and plasma lipoproteins(Ross, 1993). Macrophage cholesterol accumulation and foam cellformation are the indications of early atherogenesis with most of thecholesterol in these cells derived from plasma low-density lipoproteins(LDL). The most studied modification of LDL with a potentialpathological significance is LDL oxidation (Steinberg et al., 1989). Theinvolvement of oxidized LDL in atherosclerosis is suggested from itspresence in the atherosclerotic lesion in human and of theapolipoprotein E deficient (E⁰) mice (Yla-Herttula et al., 1989; Aviramet al., 1995), from the increased susceptibility to oxidation of LDLderived from atherosclerotic patients and also from theanti-atherogenecity of several dietary antioxidants (Steinberg et al.,1992; Frankel et al., 1993; Aviram, 1996).

High-density lipoproteins (HDL) are associated with anti-atherogenicactivity and HDL levels are inversely related to the risk of developingatherosclerosis. Paraoxonase, an enzyme, physically associated in serumwith HDL, has been shown to be inversely related to the risks ofatherogenesis (Watson et al., 1995; Aviram, 1999). The LDL oxidationhypothesis of atherosclerosis raised an extensive investigation into therole of antioxidants against LDL oxidation as a possible preventivetreatment for atherosclerosis. Efforts are made to identify natural foodproducts, which offer antioxidant defense against LDL oxidation.

Consumption of flavonoids in the diet has been shown to be inverselyassociated with morbidity from coronary heat disease, (Hertog et al.,1993; Knekt et al., 1996). Flavonoids extracted from red wine protectedLDL oxidation where added in-vitro (Frankel et al., 1993) andconsumption of red wine was shown to inhibit LDL oxidation ex-vivo(Kondo, 1994; Fuhrman et al., 1995).

Carotenoid consumption has been shown in previous epidemiologicalstudies to be associated with reduced cardiovascular mortality(Kohlmeier and Hasting, 1995). However, several dietary interventiontrials involving β-carotene have yielded inconclusive results (Mayne,1996). Lee et al. (1999) reported that among healthy women given aβ-carotene supplement for a limited time, no benefit or harm wasobserved regarding incidence of cancer and of cardiovascular diseases.Lower serum lycopene levels were associated with increase risk andmortality from coronary heart disease in a cross sectional study ofLithuanian and Swedish populations (Kristenson et al., 1997; Rao andAgarwal, 1999). Iribarren et al. (1997) found the xanthophylls luteinand zeaxanthin to be the carotenoid with the strongest inverseassociation with extreme carotid artery intima-medial thickening.

Cancer and the Effects of Carotenoids:

Cancer development is characterized by specific cellular transformationsfollowed by uncontrolled cell growth and invasion of the tumor site witha potential for subsequent detachment, transfer into the blood streamand metastases formation at distal site(s) (Ilyas et al., 1999). Allthese stages involve a number of cellular alterations including changesin proliferation rates, inactivation of tumor suppressor genes andinhibition of apoptosis (Goldsworthy et al., 1996; Knudsen et al., 1999;Ilyas et al., 1999).

Dietary exposures provide one of the environmental factors believed tobe significant in the etiology of a number of epithelioid cancer cases,notably oral and colon carcinomas. Cancer inhibitory properties for anumber of micronutrients with antioxidant properties have beendemonstrated in recent years mainly in experimental animal models (Jainet al., 1999), in cell culture studies (Schwartz and Shklar, 1992), andin some human studies (Schwartz et al., 1991). Epidemiological evidencelinks nutrition rich in vegetables and fruits, with reduced risks ofdegenerative disease, the evidence is particular compelling for cancer(Block et al., 1992). Epidemiological studies suggest that the incidenceof human cancer is inversely correlated with the dietary intake ofcarotenoids and their concentration in plasma (Ziegler, 1988). A varietyof carotenoids are present in commonly eaten foods and these compoundsaccumulate in tissues and blood plasma. Animal studies and cultured cellstudies have shown that many carotenoids such as α-carotene,β-cryptoxanthin, astaxanthin and lycopene have anticarcinogenicactivities. (Murakoshi et al., 1992; Tanaka et al., 1995; Levy et al.,1995). However, there have been contradictory reports concerning the useof β-carotene for cancer prevention (Hannekens et al., 1996). Amulticenter case-control study to evaluate the relation betweenantioxidant status and cancer has shown that lycopene but notβ-carotene, contribute to the protective effect of vegetable consumption(Kohlmeier et al., 1997).

The putative Biological Mechanisms of Cancer Inhibition of theAntioxidant Micronutrients are:

(1) Enhancement of production of cytotoxic immune cells and productionof cytokines (Schwartz et al., 1990).

(2) Activation of cancer suppressor genes such as wild p53 (Schwartz etal., 1993), or deactivation of oncogenes such as Ha-ras and mutated p53(Schwartz et al., 1992).

(3) Inhibition of angiogenesis-stimulating factors involved with tumorangiogenesis (Schwartz and Shklar, 1997).

Primary prevention or drug-based therapeutics of oral and colon canceris a public health goal but still not feasible despite major advances inunderstanding of the mechanisms at the genetic, germline, somatic,immunologic and angiogenic levels. Therefore, a great interest inpreventive nutrition has arisen focusing on the role of dietarycomponents with antioxidant activity such as several vitamins andcarotenoids, to prevent cancer (Weisburger, 1999).

Oral Cancer:

The frequency of oral cancer is 4-5% of all cancer cases in the westernworld. Squamous cell carcinoma (SCC) make up 95% of oral cancer cases.Risk factors in oral cancer include tobacco as a major risk factor, andalcohol abuse, especially when used in combination with tobacco (DeStefani et al., 1998; Hart et al., 1999; Schildt et al., 1998; Dammer etal., 1998; Bundgaard et al., 1995). Viral Infections, particularly withseveral species of Human Papilloma Virus (HPV) have been associated withboth benign and malignant oral lesions (Smith et al., 1998).

Leukoplakia is the most common pre-neoplastic condition. Leukoplakiapresents as white lesions on the oral mucosa, while erythroleukoplakiais a variant of leukoplakia in which the clinical presentation includeserythematous area as well. When biopsied, leukoplakia may show aspectrum of histologic changes ranging from hyperkeratosis, dysplasia tocarcinoma-in-situ or even invasive carcinoma. Dysplastic changes aremore frequent in erythroleokoplakia. Leukoplakia is considered apre-neoplastic lesion, which carries a 15% risk for malignanttransformation over time if dysplasia is not diagnosed in the initialbiopsy, and up to 36% transformation for lesions with dysplasia at thetime of first biopsy (Mao, 1997). Leukoplakia is associated with the useof tobacco in the majority of cases, but cases of leukoplakia innon-smoking women, have a higher risk. When leukoplakia is diagnosed,the treatment protocol consists of cessation of risk habits, andfrequent follow-up, including repeated biopsies. No effective long-termpreventive treatment is yet available.

Ki67, PCNA, CyclinD1, p53, p16, and p21 are all cell cycle associatedproteins, which are over-expressed in oral cancer and pre-cancer, andare associated with a negative prognosis in cancer cases (Schoelch etal., 1999; Yao et al., 1999; Birchall et al., 1999).

The Role of Carotenoids in the Prevention of Oral Cancer:

Vitamin A and its derivatives, by way of systemic administration ortopical application have been shown to be beneficial in regressingleukoplakia. In cases of oral cancer, vitamin-A and its derivatives havebeen shown to reduce the risk of secondary cancer (Hong et al., 1990;Gravis et al., 1999). However, in long term use they are associated withsignificant side effects, and the lesions tend to recur when treatmentis discontinued. Beta-carotenes are not associated with significant sideeffects, and there is evidence from experimental studies that indicatethey may be effective in inhibiting malignant transformation, however,there is contradictory data regarding their efficiency in clinical usefor oral cancer and pre-cancer (Stich et al., 1998). A recent study hasshown significantly lower levels of serum β-carotene and of tissueβ-carotene in smokers, which are at risk for developing oral cancer(Cowan et al., 1999).

The prognosis of oral cancer is generally poor. The mean five-yearsurvival of oral cancer cases is only about 50%, and although muchimproved diagnostic and treatment tools have been introduced, survivalhas not improved over the last two decades.

Treatment consists of surgery radiation and chemotherapy, and in mostcases is associated with severe effects on the quality of life, such asimpaired esthetics, mastication, and speech.

In view of the poor prognosis of oral cancer, prevention and regressionat the pre-malignant stage is of enormous importance. However when apre-malignant lesion such as leukoplakia is identified, very fewefficient treatment modalities are yet available for routine practice.Therefore, a continuing effort is necessary to identify new compoundsthat will be able to regress existing lesions and prevent theirtransformation into malignancy, with minimal or no side effects, toallow for long term use in patients at risk. It is also important tofind chemopreventing agents that will reduce the risk for secondarycancer in patients with primary oral cancer, which is as high as 36%.

Colon Cancer:

Colon cancer is the third most common form of cancer and the overallestimated new cases per year worldwide represent about 10% of all newcancer cases. The disease is most frequent in Occidental countriesincluding Israel. Epidemiological studies have emphasized the major roleof diet in the ethiology of colon cancer. Attempts to identify causativeor protective factors in epidemiological and experimental studies haveled to some discrepancies. Nonetheless, prospects for colorectal cancercontrol are bright and a number of possible approaches could provefruitful. Bras and associates (1999) have recently demonstrated that infamilial adenomatous polyposis patients, a population highly prone todevelop colorectal cancer, exhibit an imbalance in thepro-oxidant/antioxidant status. In addition, the levels of ascorbate andtocopherol were considerably lower in this population. Collins et al.(1998) have shown in populations from five different European countriesthat the mean 8-oxodeoxyguanosine (8-oxo-dG) concentrations inlymphocyte DNA showed a significant positive correlation with colorectalcancer. It would appear that patients with colonic cancer undergo asignificant reduction in their antioxidant reserve compared to healthysubjects. These studies support the notion that one approach to identifyprotective factors in colorectal canter will be those that provide abalanced oxidative status, or fit the antioxidant hypothesis. Thishypothesis proposes that vitamin C, vitamin E, and carotenoids occurringin fruits and vegetables afford protection against cancer by preventingoxidative damage to lipids and to DNA.

The Role of Carotenoids in the Prevention of Colon Cancer:

Recent studies suggest a protective effect of carotenoids andantioxidants, lycopene and lycopene-rich tomatoes against variouscancers, among them, colon cancer.

Rats with induced colon cancer fed lycopene or tomato juice/watersolution, had shown a lower colon cancer incidence than the controlgroup. The protective effect against colon preneoplasia associated withenhanced antioxidant properties was observed in a study where rats wereadministered a carcinogen and administered lycopene in the form of 6%oleoresin supplementation (Jain et al., 1999). Chemoprevention bylycopene of mouse lung neoplasia has also been reported (Kim et al.,1997). Kim et al. (1988) assessed the effect of carotenoids, such asfucoxanthin, lutein and phenolics such curcumin and its derivativetetrahydrocurcumin (THC) on colon cancer development in mice.Flucoxanthin, lutein, carcumin and THC significantly decreased thenumber of aberrant crypt foci compared to the control group.Proliferation rate was lower following this treatment, with highereffectiveness seen by THC. A similar effect was reported by Narisawa andassociates (1996) with the exception for β-carotene.

Human studies conducted by Pappalardo et al., (1997), compared thestatus of carotenoids in tissue and plasma in healthy subjects andsubjects with pre-cancer and cancerous lesions. The cancer subjects hadlower levels of carotenoid than those of healthy subjects.

Genetic and Breeding of Red Pepper:

Red pepper is one of the richest sources of carotenoids among vegetablecrops. Most of the domesticated varieties of red pepper belong to thespecies Capsicum annuum; pepper breeding has focused and evolved mainlyon the development of cultivars and varieties suited for use as avegetable, spice condiment, ornamental or medicinal plant. Few studieshave been devoted to the improvement of the chemical and nutritionalcomposition of peppers (Bosland, 1993; Poulos, 1994). Capsanthin is thepredominant carotenoid of the red pepper fruit and its content iscontrolled by major genes and polygenes; several genes have beenidentified along its biosynthetic pathway (Lefebvre, 1998).

Carotenoids from Red Pepper Fruits:

Red pepper fruits, especially from paprika cultivars are used in theform of powders and oleoresins as food colorants. These products arevery rich in carotenoids, some of them specific to pepper fruits. Theketo carotenoid, capsanthin, occur only in red pepper, represents 50%the carotenoids in the vegetable and contribute to the red color.Zeaxanthin and lutein, β-carotene and β-cryptoxanthin are the additionalcarotenoids found in red pepper at concentrations of 20%, 10% and 5%,respectively (Levy et al., 1995). Capsanthin accounts for 30-60% oftotal carotenoids in fully ripe fruits. The capsanthin is esterifiedwith fatty acids (nonesterified 20%; monoesterified 20-30%; diesterified40-50%). The fatty acids of esterified capsanthins are chiefly lauric(12:0), myristic (14:0) and palmitic (16:0) acid.

Increasing the carotenoid concentration in high-pigment fruits of redpepper by genetic manipulation seems to improve not only the quality ofthe fruit as a food colorant but also as an important source ofcarotenoids, particularly, capsanthin. It was found that the breedingline 4126 contains about 240 mg carotenoids/100 grams fresh weight ofwhich 120 mg are capsanthin (Levy et al., 1995). Tomatoes contain about5 mg lycopene/100 grams fresh weight, and only in special breedinglines, levels of 15 mg lycopene/100 grams fresh weight are achieved.These enormous differences in carotenoid content emphasizes the highpotential of red pepper cultivars as an appropriate food source withhigh carotenoid concentration.

Bioavailability of Carotenoids:

As a result of their lipophilic nature, carotenoids are often foundcomplexed in the food matrix with proteins, lipids and or fiber. Severalsteps are necessary for carotenoid absorption to occur. The food matrixmust be digested and the carotenoids must be released, physically andbiochemically, and combined with lipids and bile salts to form micelles.The micelles must move to the intestinal brush border membrane forabsorption and be transported into the enterocyte for subsequentprocessing. The chylomicrons move to the liver and are transported bylipoproteins for distribution to the different organs. Part of thecarotenoids in chylomicrons remnants are taken up by extra-hepatictissues before hepatic uptake (Lee et al., 1999). Thus, many factorsinfluence absorption and hence bioavailability of dietary carotenoids.Humans absorb a variety of carotenoids intact, and some carotenoidssuch, as β-carotene, β-cryptoxanthin and α-carotene can contribute tothe vitamin A status of the individual (Olson, 1999). Mathews-Roth etal. (1990) studied the absorption and distribution of (¹⁴C)canthaxanthin, a typical xanthophyll, and (¹⁴C) lycopene, an acyclichydrocarbon carotenoid, in rats and rhesus monkeys. They showed that theliver accumulated the largest amount of both, however clearance oflycopene was much slower than canthaxanthin. Stahl and Sies (1992)showed that the lycopene concentration in human plasma was increased bythe consumption of heat-processed tomato juice. Recently it was found inhumans that in a single ingestion of paprika juice containing 34.2 μmolecapsanthin and a week later tomato soup, containing 186.3 μmolelycopene, resulted in elevation of plasma carotenoids from both sources.The plasma contain only deesterified carotenoids includingnon-esterified capsanthin. The results also show that capsanthindisappear from the plasma more rapidly than lycopene (Oshima et al.,1997). Rainbow trout were fed diet supplemented with canthaxanthin andoleoresin paprika. Canthaxanthin was more efficient absorbed in theflesh of rainbow trout than paprika carotenoids (Akhtar et al., 1999).

Bioavailability of Carotenoids Esterified with Fatty Acids:

The bioavailability of paprika carotenoids in human and animal wereshown to be lower than β-carotene or canthaxanthin (Akhtar et al.,1999). One of the reasons to this reduced absorption seems to occurbecause most of the carotenoids are in an ester form with fatty acids.It is shown herein that pancreatic lipase catalyze the deesterificationof paprika carotenoids to a very limited extent. This could explain thelow bioavailability of carotenoids from paprika in animals.

Thus although the red pepper fruit is the richest in carotenoids of allother sources, the bioavailability of red pepper carotenoids is poorbecause red pepper carotenoids are esterified with fatty acids, whichprevent their efficient uptake in the gut.

Enzymatic Treatment of Esterified Carotenoids:

Several studies have indicated that esterified carotenoids may besubstrates for enzymatic hydrolysis. Japanese Laid-Open Patent No.59-91155 to Masahiro et al. teaches a method for manufacture of astable, odorless pigment from natural carotenoid-containing materials bytransesterification. The authors demonstrated that moleculardistillation, using a thin-film or falling film apparatus, may be usedto purify carotenoids following the chemical conversion of odiferousunsaturated fatty acid residues of oleoresin to saturated fatty acids,and that a lipase from Candida cylindracea (renamed Candida rugosa) canbe useful as an auxilliary agent in the transesterification. Althoughthe resultant transesterified, fatty acid-containing carotenoid pigmentsdisclosed are more stable, and less odiferous than the native pigments,the production of free carotenoids is not mentioned.

Lipase is known to be important in the in-vivo hydrolysis of esters ofVitamin A (Harrison, J Nutr 2000;130:340S-344S). Similarly, lipase hasbeen used in the synthesis of Vitamin A, in the acylation andesterification of Vitamin A precursors (U.S. Pat. No. 5,902,738 to Orsatet al; Maugard et al, Biotechnol Prog 2000;16:358-362), and in theesterification of the carotenoid (3R, 3′R, 6′R) lutein to (3R, 3′R)zeaxanthin (Khachik F, J Nat Prod; 2003:66:67-70).

In an attempt to identify the key lipases responsible for metabolism ofcarotenoids in the gut, Breithaupt et al. (Compar. Biochem and PhysiolPart B 2002;132:721-28) compared the substrate specificity of a numberof lipases towards various red and yellow carotenoids and derivatives(Vitamin A). They found that whereas the retinyl palmitate was readilyhydrolyzed by pancreatic lipase, carotenoid diesters were poorsubstrates for pancreatic lipase and Candida rugosa lipase. Results withcholesterol esterase were better, but no quantitative deesterification,similar to the results with retinyl palmitate, was observed.

Breithaupt (Breithaupt, D E, et al, Z. Naturforsch 2000;55:971-75) andKhachik (Khachik F, et al, Anal Chem 1997;69:1873-81) describe partialhydrolysis of natural carotenoids using lipase. Khachik et al. usedlipase for analysis of the carotenoid and carotenoid metabolites inhuman milk and serum. Breithaupt used C. rugosa lipase for enzymatictreatment of natural esterified red and green pepper carotenoids, butwas only partially successful, producing a preparation comprising amixture of deesterified and esterified carotenoids, as analyzed by HPLC.Similar inability to demonstrate efficient, quantitative enzymatichydrolysis of natural carotenoids had been reported using Pseudomonasfluorescens cholesterol esterase (Jacobs, et al, Comp Biochem Physiol1982;72B: 157-160). In describing his failure to reduce thecontamination of the carotenoid product with mono- and diesterifiedcarotenoids, Breithaupt hypothesized that the persistence of mono- anddiesterified derivatives of the red-pepper carotenoid capsanthin was dueto substrate specificity of the lipase, concluding that “neitherdiesterified nor monoesterified carotenoids are preferred substrates”(Breithaupt, D E Z. Naturforsch 55c; page 974, right column to page 975,left column).

There is thus a widely recognized need for, and it would be highlyadvantageous to have, a method of efficient deesterification ofesterified carotenoids devoid of the limitations inherent in chemicalsaponification, so as to render such carotenoids bioavailable to humanand animal.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of extracting red pepper oleoresin, the method comprisinghomogenizing red-pepper fruits in water into a juice; centrifuging thejuice so as to obtain a pellet; mixing the pellet with ethanol and ethylacetate; homogenizing the pellet with the ethanol and the ethyl acetate;removing dry material; and evaporating solvents so as to obtain redpepper oleoresin.

According to further features in preferred embodiments of the inventiondescribed below, a weight ratio between the red-pepper fruits and thewater is 80-120 parts of fruit to 20-60 parts of water.

According to still further features in the described preferredembodiments the red-pepper fruits are frozen.

According to still further features in the described preferredembodiments the red-pepper fruits are fresh.

According to still further features in the described preferredembodiments the juice is centrifuged at 20,000-30,000 g for 10-30minutes.

According to still further features in the described preferredembodiments the pellet is mixed with 1-3 parts of the ethanol and 5-15parts of the ethyl acetate.

According to still further features in the described preferredembodiments removing the dry material is by centrifugation.

According to still further features in the described preferredembodiments evaporating the solvents is at 40-50° C.

According to still further features in the described preferredembodiments evaporating the solvents is under vacuum.

According to another aspect of the present invention there is provided amethod of determining an efficiency of an esterase in increasing afraction of free carotenoids in a source of carotenoids in which atleast some of the carotenoids are fatty acid esterified carotenoids, themethod comprising contacting the source of carotenoids with the esteraseunder preselected experimental conditions; and using a carotenoidsdetection assay for determining the efficiency of the esterase inincreasing the fraction of the free carotenoids in the source ofcarotenoids.

According to still another aspect of the present invention there isprovided a method of screening for esterases efficient in increasing afraction of free carotenoids in a source of carotenoids in which atleast some of the carotenoids are fatty acid esterified carotenoids, themethod comprising contacting the source of carotenoids separately witheach of the esterases under preselected experimental conditions; andusing a carotenoids detection assay for determining the efficiency ofeach of the esterases in increasing the fraction of the free carotenoidsin the source of carotenoids, thereby screening for esterases efficientin increasing the fraction of free carotenoids in the source ofcarotenoids.

According to yet another aspect of the present invention there isprovided a method of optimizing reaction conditions for increasing afraction of free carotenoids in a source of carotenoids in which atleast some of the carotenoids are fatty acid esterified carotenoids, viaan esterase, the method comprising contacting the source of carotenoidswith the esterase under different preselected experimental conditions;and using a carotenoids detection assay for determining the efficiencyof the esterase in increasing the fraction of the free carotenoids inthe source of carotenoids under each of the different preselectedexperimental conditions, thereby optimizing the reaction conditions forincreasing the fraction of free carotenoids in the source of carotenoidsin which at least some of the carotenoids are fatty acid esterifiedcarotenoids via the esterase.

According to still another aspect of the present invention there isprovided a method of increasing a fraction of free carotenoids in asource of carotenoids in which at least some of the carotenoids arefatty acid esterified carotenoids, the method comprising contacting thesource of carotenoids with an effective amount of an esterase underconditions effective in deesterifying the fatty acid esterifiedcarotenoids, thereby increasing the fraction of free carotenoids in thesource of carotenoids.

According to yet another aspect of the present invention there isprovided a method of increasing a fraction of free carotenoids in asource of carotenoids in which at least some of the carotenoids arefatty acid esterified carotenoids, the method comprising contacting thesource of carotenoids with an effective amount of an esterase underconditions effective in deesterifying the fatty acid esterifiedcarotenoids, wherein the conditions effective in deesterifying the fattyacid esterified carotenoids are characterized by addition of at leastone additive selected from the group consisting of a cellulose degradingenzyme, a protein degrading enzyme, a pectin degrading enzyme, anemulsifier; and at least one metal ion, thereby increasing the fractionof free carotenoids in the source of carotenoids.

According to still another aspect of the present invention there isprovided a method of increasing a fraction of free carotenoids in asource of carotenoids in which at least some of the carotenoids arefatty acid esterified carotenoids, the method comprising contacting thesource of carotenoids with an effective amount of an esterase underconditions effective in deesterifying the fatty acid esterifiedcarotenoids, so as to produce a source of at least partiallydeesterified carotenoids, and extracting said source of at leastpartially deesterified carotenoids with ethyl acetate under alkalineconditions, thereby increasing the fraction of free carotenoids in thesource of carotenoids.

According to another aspect of the present invention there is provided amethod of increasing a fraction of free carotenoids in a source ofcarotenoids in which at least some of the carotenoids are fatty acidesterified carotenoids, the method comprising contacting the source ofcarotenoids with an effective amount of an immobilized esterase underconditions effective in deesterifying the fatty acid esterifiedcarotenoids, thereby increasing the fraction of free carotenoids in thesource of carotenoids.

According to still another aspect of the present invention there isprovided a method of increasing a fraction of free carotenoids in asource of carotenoids in which at least some of the carotenoids arefatty acid esterified carotenoids, the method comprising contacting thesource of carotenoids with an effective amount of an esterase and arecycled emulsifier under conditions effective in deesterifying thefatty acid esterified carotenoids, thereby increasing the fraction offree carotenoids in the source of carotenoids.

According to yet another aspect of the present invention there isprovided a method of reducing a fraction of Vitamin E in a source ofcarotenoids in which at least some of the carotenoids are fatty acidesterified carotenoids, the method comprising contacting the source ofcarotenoids with an effective amount of an esterase under conditionseffective in deesterifying the fatty acid esterified carotenoids, so asto produce a source of at least partially deesterified carotenoids, andchromatographically extracting the fraction of Vitamin E away from saidsource of at least partially deesterified carotenoids, thereby reducingthe fraction of Vitamin E in the source of carotenoids.

According to further features in preferred embodiments of the inventiondescribed below, the method further comprising extracting freecarotenoids from the source of carotenoids.

According to an additional aspect of the present invention there isprovided a source of carotenoids having an increased fraction of freecarotenoids and produced by the method described herein.

According to an additional aspect of the present invention there isprovided a food additive comprising the source of carotenoids having anincreased fraction of free carotenoids as described herein.

According to an additional aspect of the present invention there isprovided a feed additive comprising the source of carotenoids having anincreased fraction of free carotenoids as described herein.

According to still another aspect of the present invention there isprovided a composition of matter comprising enzymatically deesterifiedred carotenoids, the composition of matter characterized by at leastabout 40 percent by weight capsanthin, at least about 15 percent byweight zeaxanthin and capsolutein, at least about 2 percent by weightviolaxanthin, at least about 1 percent by weight capsorubin, at leastabout 5 percent by weight beta cryptoxanthin and at least about 3percent by weight beta carotene, and wherein said composition of matteris characterized by antioxidant activity, as measured by lipidoxidation.

According to further features in preferred embodiments of the inventiondescribed below, the source of carotenoids is characterized in that amajority of the carotenoids in the source of carotenoids are the fattyacid esterified carotenoids.

According to still further features in the described preferredembodiments the source of carotenoids is red pepper.

According to still further features in the described preferredembodiments the source of carotenoids is red pepper powder.

According to still further features in the described preferredembodiments the source of carotenoids is paprika.

According to still further features in the described preferredembodiments the source of carotenoids is red pepper oil extract.

According to still further features in the described preferredembodiments the source of carotenoids is red pepper oleoresin.

According to still further features in the described preferredembodiments the source of carotenoids is selected from the groupconsisting of apple, apricot, avocado, blood orange cape gooseberry,carambola, chilli, clementine, kumquat, loquat, mango, minneola,nectarine, orange, papaya, peach, persimmon, plum, potato, pumpkin,tangerine and zucchini.

According to still further features in the described preferredembodiments the esterase is selected from the group consisting of alipase, a carboxyl ester esterase and a chlorophylase, preferably alipase.

According to still further features in the described preferredembodiments the lipase is selected from the group consisting ofbacterial lipase, yeast lipase, mold lipase and animal lipase.

According to still further features in the described preferredembodiments the esterase is an immobilized esterase.

According to still further features in the described preferredembodiments the preselected experimental conditions, the differentpreselected experimental conditions and/or the conditions effective indeesterifying the fatty acid esterified carotenoids, comprise at leastone of addition of a cellulose degrading enzyme; addition of a pectindegrading enzyme; addition of an emulsifier; and addition of at leastone metal ion.

According to still further features in the described preferredembodiments the at least one metal ion is selected from the groupconsisting of Ca⁺⁺ and Na⁺.

According to still further features in the described preferredembodiments the addition of the at least one metal ion is by addition ofat least one salt of said metal ion.

According to still further features in the described preferredembodiments the at least one salt is selected from the group consistingof CaCl₂ and NaCl.

According to still further features in the described preferredembodiments the cellulose degrading enzyme is selected from the groupconsisting of C1 type beta-1,4 glucanase, exo-beta-1,4 glucanase,endo-beta-1,4 glucanase and beta-glucosidase.

According to still further features in the described preferredembodiments the proteins degrading enzyme is selected from the groupconsisting of tripsin, papain, chymotripsins, ficin, bromelin,cathepsins and rennin.

According to still further features in the described preferredembodiments the pectin degrading enzyme is selected from the groupconsisting of a pectin esterase, pectate lyase and a polygalacturonase.

According to still further features in the described preferredembodiments the emulsifier is a non-ester emulsifier. According to stillfurther features in the described preferred embodiments the emulsifieris lecithin.

According to still further features in the described preferredembodiments the emulsifier is deoxycholate.

According to still further features in the described preferredembodiments the emulsifier is a non-ionic detergent, such as, but notlimited to, polyoxyethylensorbitane monolaurate (TWEEN-20).

According to still further features in the described preferredembodiments the emulsifier is derived from bile, gum—Arabic or sodiumsalt of free fatty acids.

According to yet further features in the described preferred embodimentsthe emulsifier is a recycled emulsifier.

According to still further features in the described preferredembodiments the carotenoids detection assay is a chromatography assay.

According to still further features in the described preferredembodiments the chromatography assay is selected from the groupconsisting of thin layer chromatography and high performance liquidchromatography.

According to still another aspect of the present invention there isprovided an article of manufacture comprising a packaging material andat least one antioxidant unit dosage, the antioxidant unit dosagecomprising a composition of matter comprising at least about 40 percentby weight capsanthin, at least about 15 percent by weight zeaxanthin andcapsolutein, at least about 2 percent by weight violaxanthin, at leastabout 1 percent by weight capsorubin, at least about 5 percent by weightbeta cryptoxanthin, at least about 3 percent by weight beta carotene andat least 10 mg per gram Vitamin E and a pharmaceutically acceptablecarrier in each single unit dosage, and wherein the packaging materialcomprises a label or package insert indicating that the composition ofmatter is for increasing antioxidant levels in a subject.

According to further features in the described preferred embodiments thearticle of manufacture of comprises about 20 mg per gram Vitamin E.

According to still further features in the described preferredembodiments the composition of matter further comprises apharmaceutically acceptable excipient selected from the group consistingof carboxymethylcellulose, microcrystalline cellulose, starch, andmodified starch.

According to yet further features in the described preferred embodimentsthe antioxidant unit dosage is designed for oral administration.

According to still further features in the described preferredembodiments the antioxidant unit dosage is selected from the groupconsisting of a tablet, a caplet, and a capsule.

According to further features in the described preferred embodiments thecomposition of matter is in the form of a liquid dosage form.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing methods of determining anefficiency of an esterase in increasing a fraction of free carotenoidsin a source of carotenoids in which at least some of the carotenoids arefatty acid esterified carotenoids; screening for esterases efficient inincreasing a fraction of free carotenoids in a source of carotenoids inwhich at least some of the carotenoids are fatty acid esterifiedcarotenoids; optimizing reaction conditions for increasing a fraction offree carotenoids in a source of carotenoids in which at least some ofthe carotenoids are fatty acid esterified carotenoids, via an esterase;and increasing a fraction of free carotenoids in a source of carotenoidsin which at least some of the carotenoids are fatty acid esterifiedcarotenoids; and a source of carotenoids having an increased fraction offree carotenoids, which can serve as a food and/or feed additive; and arich source from which one can extract to purification desiredcarotenoids.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice In the drawings:

FIG. 1 is a HPLC chromatogram of natural red pepper carotenoids(obtained from oleoresin);

FIG. 2 is a HPLC chromatogram of natural red pepper (paprika)carotenoids following chemical saponification, the chromatogram containsmostly about 9 peaks of: (i) capsanthin (6.1 min); (ii) violaxanthin(7.36 min); (iii) capsanthin (8.89 min); (iv) cis-capsanthin (10.33);(v) capsolutein (10.83 min); (vi) Zeaxanthin (11.2 min); (vii)cis-Zeaxanthin (12.0 min); (viii) β-crypotxanthin (14.36 min); and (ix)β-carotene;

FIG. 3 is a HPLC chromatogram of natural red pepper (paprika)carotenoids following treatment with pectinase, protease, cellulase andlipase in the presence of deoxycholate;

FIG. 4 is a HPLC chromatogram of paprika oleoresin carotenoids followingtreatment with deoxycholate and lipase;

FIGS. 5 a-c are HPLC chromatograms of paprika oleoresin carotenoidsfollowing treatment with varying concentarations of deoxycholate (2%, 3%and 4%, respectively) and lipase;

FIG. 6 demonstrates the steps of a method of extracting oleoresin fromred pepper fruits, according to the present invention;

FIGS. 7 a-7 c are HPLC chromatograms of paprika oleoresin carotenoidsextracted with fresh and recycled immobilized Candida rugosa lipase. 100mg equivalent matrix-bound C. rugosa lipase was used to extract 20 mg ofpaprika oleoresin in the presence of deoxycholate and water by shakingfor 24 hours at 37° C., followed by ethyl acetate extraction, asdescribe in Materials and Experimental Methods hereinbelow. FIG. 7 ashows an HPLC chromatogram following extraction of oleoresin withfreshly prepared immobilized lipase. FIGS. 7 b and 7 c show the HPLCchromatograms of fresh samples of oleoresin following extraction withrecycled immobilized lipase collected by precipitation after use. Notethe identity of HPLC profile using fresh immobilized lipase (FIG. 7 a),once recycled immobilized lipase (FIG. 7 b) and twice recycledimmobilized lipase (FIG. 7 c), indicating no significant loss of enzymeactivity;

FIG. 8 shows the retention of lipase activity in the recycledmatrix-bound lipase. Enzyme activity (expressed as percentdeesterification of paprika oleoresin as compared with fresh matrixbound lipase) remained stable with no significant loss through 3 cyclesof extraction and recovery of lipase;

FIGS. 9 a-9 d are HPLC chromatograms of paprika oleoresin carotenoidsextracted with fresh and recycled deoxycholate. 20 mg of paprikaoleoresin was extracted with immobilized lipase in the presence of 200mg fresh or recycled deoxycholate and water by shaking for 24 hours at37° C., followed by ethyl acetate extraction, as describe in Materialsand Experimental Methods hereinbelow. FIG. 9 a shows an HPLCchromatogram following extraction of oleoresin with freshly prepareddeoxycholate. FIGS. 9 b to 9 d show the HPLC chromatograms of freshsamples of oleoresin following extraction with recycled deoxycholaterecycled by dehydration of the aqueous phase by lyophyllization or ovendrying after use. Note the identity of HPLC profile using freshdeoxycholate (FIG. 9 a), once recycled deoxycholate (FIG. 9 b), twicerecycled deoxycholate (FIG. 9 c) and thrice recycled deoxycholate (FIG.9 d), indicating no significant loss, and an apparent increase, ofemulsification activity with recycling;

FIG. 10 shows the retention of emulsification activity in the recycleddeoxycholate. Enzyme activity (expressed as percent deesterification ofpaprika oleoresin as compared with freshly prepared deoxycholate)remained stable with no significant loss, and apparent increase, through3 cycles of drying and reconstitution of deoxycholate;

FIGS. 11 a and 11 b are HPLC chromatograms of de-esterified Paprika oilshowing effective Vitamin E removal by Flurasil column. DeesterifiedPaprika oil, rich in Vitamin E (retention time=5.1 minutes, FIG. 11 a)was separated on a Florisil column equilibrated with hexane and washedwith hexane to remove yellow carotenoids. Red carotenoids (xanthophylls)were eluted with ethyl acetate, and analyzed on HPLC (FIG. 11 b). Notethe greater than 40 fold reduction in Vitamin E content of the redcarotenoids after Flurasil purification (FIG. 11 b, 5.1 minutesretention time);

FIG. 12 is a Table showing the superior stability of enzymaticallydeesterified paprika oleoresin, by maintenance of color in water. Theabsorbance at 474 nm (red color) of a paprika oleoresin emulsionprepared in water (1 mg/100 ml water) containing 0.15% or 0.03% Tween-20detergent was measured spectrophotometrically. Note the superior colorstability of the deesterified oleoresin in low detergent (0.03%)conditions, over 30 days storage;

FIGS. 13 a-13 d are HPLC chromatograms of enzymatically deesterifiedpaprika oleoresin carotenoids extracted under different conditions. FIG.13 a shows a chromatogram of enzymatically deesterified paprikaoleoresin carotenoids extracted with hexane. FIG. 13 b shows achromatogram of enzymatically deesterified paprika oleoresin carotenoidsextracted with with ethyl-acetate. FIG. 13 c shows a chromatogram ofenzymatically deesterified paprika oleoresin carotenoids extracted withethyl acetate, with pH adjustment to pH 9.5, and FIG. 13 d shows achromatogram of enzymatically deesterified paprika oleoresin carotenoidsextracted with ethyl acetate, without pH adjustment. 20 mg of paprikaoleoresin containing 74 μg total carotenoids/mg oil was deesterified inthe presence of lipase, deoxycholate and water by shaking for 24 hoursat 37° C. Ethyl acetate or hexane extraction, as describe in Materialsand Experimental Methods hereinbelow, was then performed, either with(FIG. 13 c) or without (FIG. 13 d) adjustment of pH to 9.5 with NaOH.Note the identity of all of the HPLC chromatograms;

FIGS. 14 a and 14 b are Tables showing the effect of pH adjustment onthe efficiency of carotenoid and Vitamin E extraction followingenzymatic deesterification. FIG. 14 a compares the carotenoidcomposition following enzymatic deesterification of oleoresin, asdescribed hereinabove, and alkaline treatment (pH adjustment) or notreatment (unadjusted). FIG. 14 b compares the composition (mg/g oil) ofextracted carotenoids by mass. Note both the enrichment of capsanthin,Vitamin E and other carotenoids by percent, and the greatly superiorrecovery of total carotenoids and Vitamin E (FIG. 14 b) with alkalinetreatment;

FIGS. 15 a and 15 b are graphs of inhibition of lipid oxidation, showingthe superior antioxidant properties of enzymatically deesterifiedcarotenoids. Lipid oxidation (assayed by met-myoglobin-catalyzed dieneconjugation) was measured spectrophotometrically over time. Antioxidantactivity of enzymatically deesterified carotenoids (40 μM concentration)containing Vitamin E (Capsivit, closed squares) and lacking Vitamin E(Saponified carotenoids, closed triangles), was compared with that of 40μM lycopene (x) and beta-carotene (stars). Controls indicate the extentof lipid oxidation without added antioxidants (closed diamonds). Notethe superior inhibition of lipid oxidation with enzymaticallydeesterified carotenoids containing Vitamin E (Capsivit, closed squares)and lacking Vitamin E (Saponified carotenoids, closed triangles), atboth blood pH (pH 7.0, FIG. 15 a), and stomach acid pH (pH 3, FIG. 15b);

FIGS. 16 a and 16 b are bar graphs showing the superior antioxidanteffects of enzymatically deesterified carotenoids, as compared to othercarotenoid antioxidants. Note the superior inhibition of lipid oxidationby enzymatically deesterified carotenoids (Capsivit and Saponifiedcarotenoids) at 10 μM (grey squares), 20 μM (dark grey squares) and 40μM (light squares) antioxidant concentration, with nearly 6 timesgreater protection of the lipids from oxidation, compared to thebeta-carotene and lycopene, at 40 μM; and

FIG. 17 is a Table showing the composition of enzymatically deesterifiedcarotenoids containing Vitamin E and depleted of Vitamin E, inpercentage composition (wt/wt).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of methods of (i) determining an efficiency ofan esterase in increasing a fraction of free carotenoids in a source ofcarotenoids in which at least some of the carotenoids are fatty acidesterified carotenoids; (ii) screening for esterases efficient inincreasing a fraction of free carotenoids in a source of carotenoids inwhich at least some of the carotenoids are fatty acid esterifiedcarotenoids; (iii) optimizing reaction conditions for increasing afraction of free carotenoids in a source of carotenoids in which atleast some of the carotenoids are fatty acid esterified carotenoids, viaan esterase; (iv) increasing a fraction of free carotenoids in a sourceof carotenoids in which at least some of the carotenoids are fatty acidesterified carotenoids; and (iv) an efficient method of extracting redpepper oleoresin. The present invention is further of a source ofcarotenoids having an increased fraction of free carotenoids, which canserve as a food and/or feed additive and as a rich source from which toextract to substantial purification desired carotenoids.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

According to one aspect of the present invention there is provided amethod of extracting red pepper oleoresin. The red pepper fruit can beeither fresh or frozen. The method is effected by homogenizingred-pepper fruits in water into a juice; centrifuging the juice so as toobtain a pellet; mixing the pellet (either directly or after freezing)with ethanol and ethyl acetate; homogenizing the pellet with the ethanoland the ethyl acetate; removing dry material; and evaporating solventsso as to obtain red pepper oleoresin.

As is further detailed and exemplified hereinbelow, esterifiedcarotenoids can be deesterified from the pellet (directly or afterfreezing), or, preferably, from the oleoresin derived therefrom viaextraction as descried above, by a lipase preferably in the presence ofa cellulase and a pectinase.

Preferably, a weight ratio between the red-pepper fruits and the wateris 80-120 parts of fruit to 20-60 parts of water. Still preferably, thejuice is centrifuged at 20,000-30,000 g for 10-30 minutes. Yetpreferably, the pellet is mixed with 1-3 parts of the ethanol and 5-15parts of the ethyl acetate. Still preferably, removing the dry materialis by centrifugation. Preferably, evaporating the solvents is at 40-50°C. and preferably under vacuum.

According to another aspect of the present invention there is provided amethod of determining an efficiency of an esterase in increasing afraction of free carotenoids in a source of carotenoids in which atleast some of the carotenoids are fatty acid esterified carotenoids. Themethod according to this aspect of the present invention is effected bycontacting the source of carotenoids with the esterase under preselectedexperimental conditions; and using a carotenoids detection assay fordetermining the efficiency of the esterase in increasing the fraction ofthe free carotenoids in the source of carotenoids.

According to still another aspect of the present invention there isprovided a method of screening for esterases efficient in increasing afraction of free carotenoids in a source of carotenoids in which atleast some of the carotenoids are fatty acid esterified carotenoids. Themethod according to this aspect of the present invention is effected bycontacting the source of carotenoids separately with each of theesterases under preselected experimental conditions; and using acarotenoids detection assay for determining the efficiency of each ofthe esterases in increasing the fraction of the free carotenoids in thesource of carotenoids, thereby screening for esterases efficient inincreasing the fraction of free carotenoids in the source ofcarotenoids.

According to yet another aspect of the present invention there isprovided a method of optimizing reaction conditions for increasing afraction of free carotenoids in a source of carotenoids in which atleast some of the carotenoids are fatty acid esterified carotenoids, viaan esterase. The method according to this aspect of the presentinvention is effected by contacting the source of carotenoids with theesterase under different preselected experimental conditions; and usinga carotenoids detection assay for determining the efficiency of theesterase in increasing the fraction of the free carotenoids in thesource of carotenoids under each of the different preselectedexperimental conditions, thereby optimizing the reaction conditions forincreasing the fraction of free carotenoids in the source of carotenoidsin which at least some of the carotenoids are fatty acid esterifiedcarotenoids via the esterase.

Preferably, the carotenoids detection assay is a chromatography assay,such as, but not limited to, thin layer chromatography (TLC) and highperformance liquid chromatography (HPLC). These assays are well knownfor, and are frequently used in the characterization of differentcarotenoids.

According to still another aspect of the present invention there isprovided a method of increasing a fraction of free carotenoids in asource of carotenoids in which at least some of the carotenoids arefatty acid esterified carotenoids. The method according to this aspectof the present invention is effected by contacting the source ofcarotenoids with an effective amount of an esterase under conditionseffective in deesterifying the fatty acid esterified carotenoids,thereby increasing the fraction of free carotenoids in the source ofcarotenoids. Once freed, individual non-esterified carotenoids or groupsof similar non-esterified carotenoids can be extracted and purified tosubstantial homogeneity using methods well known in the art, such as,but not limited to, extraction with organic solvents followed by phaseseparation, various chromatographies, etc.

The source of carotenoids, rich in free, non-esterified carotenoids,produced by the method of the present invention, and/or the freecarotenoids further purified therefrom can be used as food and/or feedadditives in human or animal diet, to serve as natural antioxidantsand/or food, animal and cosmetic natural colorants.

A preferred source of carotenoids according to the present invention ischaracterized in that a majority of the carotenoids in the source ofcarotenoids are fatty acid esterified carotenoids, such as, for example,red pepper derived carotenoids. Red pepper is one of the richest sourcesof carotenoids among vegetable crops. Most of the domesticated varietiesof red pepper belong to the species Capsicum annuum; pepper breeding hasfocused and evolved mainly on the development of cultivars and varietiessuited for use as a vegetable, spice condiment, ornamental or medicinalplant. Few studies have been devoted to the improvement of the chemicaland nutritional composition of peppers (Bosland, 1993; Poulos, 1994).Capsanthin is the predominant carotenoid of the red pepper fruit and itscontent is controlled by major genes and polygenes; several genes havebeen identified along its biosynthetic pathway (Lefebvre, 1998).

Red pepper fruits, especially from paprika cultivars are used in theform of powders and oleoresins as food colorants. These products arevery rich in carotenoids, some of them specific to pepper fruits. Theketo carotenoid, capsanthin, occur only in red pepper, represents 50%the carotenoids in the vegetable and contribute to the red color.Zeaxanthin and lutein, β-carotene and β-cryptoxanthin are the additionalcarotenoids found in red pepper at concentrations of 20%, 10% and 5%,respectively (Levy et al., 1995). Capsanthin accounts for 30-60% oftotal carotenoids in fully ripe fruits. The capsanthin is esterifiedwith fatty acids (nonesterified 20%; monoesterified 20-30%; diesterified40-50%). The fatty acids of esterified capsanthins are chiefly lauric(12:0), myristic (14:0) and palmitic (16:0) acid. The bioavailability offatty acids esterified carotenoids is, nevertheless, very low.

Other plant species that containing fatty acid esterified carotenoids,including, but not limited to, apple, apricot, avocado, blood orangecape gooseberry, carambola, chilli, clementine, kumquat, loquat, mango,minneola, nectarine, orange, papaya, peach, persimmon, plum, potato,pumpkin, tangerine and zucchini, can also be used as a source ofcarotenoids for the present invention. The esterified carotenoidscontent of these fruits are described in Dietmar E. Breithaupt andAmeneh Bamedi “Carotenoid ester in vegetables and fruits: A screeningwith emphasis on beta-cryptoxanthin esters” J. Agric. Food Chem. 2001,49, 2064-2070, which is incorporated herein by reference.

Any type of esterase that can deesterify fatty acid esterifiedcarotenoids can be used to implement the present invention. Methods forscreening for most efficient esterases and suitable conditions for theiractivity with respect to different types of substrates (carotenoidssources) are also described herein. The esterase of choice can be, forexample, a lipase, a carboxyl ester esterase or a chlorophylase,preferably a lipase. Enzymes species belonging to these families areknown to deesterify a wide range of fatty acid esters, i.e., to have awide range of substrate specificity. Different lipases can be used inthe method of the present invention, including, for example, thoseobtained from bacterial, yeast or animal sources. The esterase usedwhile implementing the methods of the present invention can be free insolution or, in order to improve the accessibility to the esterase andits re-use, it can be immobilized on various carrier materials. Methodsfor immobilization of esterases on solid matrices, their use incatalytic rections, recovery and reuse are described extensively in theliterature (see, for example, U.S. Patent Application No. 5,902,738 toOrsat, et al., which is incorporated herein by reference). Briefly, theimmobilization can be effected covalently or non-covalently, preferablynon-covalently, by simple adsorption on a suitable carrier materialhaving a large surface. Since esterase and carrier material areinsoluble in organic solvents, no measurable desorption takes placeduring the reaction. Suitable carrier materials are many of the usual,inexpensive filter aids, adsorbents, ion exchangers and chromatographymaterials, such as Florisil.RTM., diatomaceous earth, bentonite,cellulose, molecular sieve, Amberlite.RTM., Amberlyst.RTM., silica gelor aluminum oxide and the like, as well as other inexpensive materialshaving large surface areas, such as sand, sintered glass or hollowfibres and the like. Alternatively, commercially available, alreadyimmobilized esterase preparations preparations can also be used, forexample the lipase preparations from Meito Sangyo and BoehringerMannheim GmbH: (Lipase PLC: Lipase PL immobilized on diatomaceous earth;Lipase PLG: Lipase PL immobilized on granulated diatomaceous earth;Lipase L-2 (Chirazyme.RTM. L-2, formerly Novozym.RTM. SP 435, RocheDiagnostics, GmbH, Mannheim, Germany): lipase from Candida antarctica,immobilized on macroporous polyacryl. In one preferred embodiment theimmobilized lipase is Candida rugosa lipase immobilized on porousacrylic beads (Cat # L1150, Sigma Chemicals, St Louis Mo.).

If desired, the immobilization of the esterase can also be effected inthe presence of a “cholanic salt” emulsifier (co-immobilization), bymeans of which the activity can in part be controlled (activator).Suitable cholanic salts are e.g. sodium cholate and sodium deoxycholate.As is further detailed below, an oil-in-water or preferably water-in-oilemulsion of the carotenoid source is prepared in order to enhancecatalytic activity of the esterase employed. Other means to enhanceenzyme activity can also be practiced, as described hereinbelow.

While reducing the present invention to practice, it was uncovered thatmatrix-bound, immobilized esterases can be recovered from thedeesterification reaction mixture, and reused numerous times (seeExample 5 hereinbelow). Surprisingly, deesterification efficiency, asmeasured by comparison of HPLC profiles of the resultant carotenoidfractions, was not significantly affected by using recycled,matrix-bound immobilized lipase (see FIGS. 7 a-7 c, hereinbelow),retaining greater than 95% original activity. The recovery and reuse ofrecycled lipase for deesterification of carotenoids is of greatadvantage not only for the improved simplicity and reduction of coststhat it affords, but also for the greatly superior purity of effluentwastes from the deesterification process, which is of crucialenvironmental and ecological concern.

Thus, according to the present invention, there is provided a method ofincreasing a fraction of free carotenoids in a source of carotenoids inwhich at least some of the carotenoids are fatty acid esterifiedcarotenoids, the method comprising contacting the source of carotenoidswith an effective amount of an immobilized esterase under conditionseffective in deesterifying the fatty acid esterified carotenoids,thereby increasing the fraction of free carotenoids in the source ofcarotenoids.

According to one preferred embodiment, the immobilized esterase isselected from the group consisting of an immobilized lipase, animmobilized carboxyl ester esterase and an immobilized chlorophylase. Inanother, more preferred embodiment, the immobilized esterase is animmobilized lipase, for example, Candida rugosa lipase. The immobilizedlipase can also be an immobilized bacterial lipase, immobilized yeastlipase, immobilized mold lipase and/or immobilized animal lipase. In amost preferred embodiment the immobilized lipase is a recycledimmobilized lipase, as dscribed in detail hereinbelow.

It will be noted, that the deesterification processes for preparingenzymatically deesterified carotenoids may be carried out in batch mode,or in continuous flow cocurrent or counter-current mode. A continuousprocess can use a reactor containing enzyme catalyst, said reactor beinga packed bed, fluidized bed or ebullating bed where the enzyme catalystremains in the bed and reactants continuously flow through the bed.Batch and continuous processes may be carried out in a singledeesterification step or multiple steps. The use of multiple stepspermits use of lower enzyme-oleoresin ratios in each step, but requiresmultiple separation steps. Single step batch or cocurrent continuousprocesses require relatively high ratios of enzyme to oleoresin in theinitial reaction mixture, but are generally more economical thanmulti-step processes. Continuous flow reactors suitable for lipasereactions are described in detail in, for example, U.S. Pat. No.5,288,619 to Brown et al., incorporated herein by reference.

Lipases typically catalyze the deesterification of triglycerides anddiglycerides containing fatty acids bond to glycerol by ester bond. Thecarotenoids in, for example, paprika are esterified by fatty acids suchas myristic, lauric, palmitic stearic, oleic and linoleic acids and forthis reason they are different from triglycerides which are the naturalsubstrates for lipases. Lipases are known to hydrolyze emulsified acyllipids, as they are active on a water/lipid interface. For this reason,deoxycholate improves the reaction of the enzyme and its concentrationis important to receive a high reactivity of the enzymes. Lipasecatalyzed reactions are accelerated by Ca²⁺ ions since the freed fattyacids are precipitated as insoluble Ca-salts. Introduction of Ca²⁺ ionsin the process described herein enhances deesterification.

Thus, according to preferred embodiments of the present invention, thepreselected experimental conditions, the different preselectedexperimental conditions and/or the conditions effective in deesterifyingthe fatty acid esterified carotenoids, comprise, for example, theaddition of a cellulose degrading enzyme; the addition of a proteinsdegrading enzyme; the addition of a pectin degrading enzyme; theaddition of an emulsifier to the reaction mixture; and/or the additionof at least one metal ion to the reaction mixture, e.g., the addition ofsalts, such as CaCl₂ and/or NaCl. Other reaction conditions such as theaddition of effectors, temperature, pH, etc., can also be optimized foreach combination of enzyme and substrate.

The degrading enzymes used in context of the present invention serve todegrade their respective substrates present in the reaction mixture inorder to avoid sequestering effects and reduce the viscosity of thereaction mixture.

The cellulose of choice can be a C₁ type beta-1,4 glucanase,exo-beta-1,4 glucanase, endo-beta-1,4 glucanase and/or beta-glucosidasefrom plant, insect or bacterial source. The proteins degrading enzymecan be, for example, tripsin, papain, chymotripsins, ficin, bromelin,cathepsins and/or rennin. The type and amount of the proteins degradingenzyme is controlled so as to avoid degradation of the esterase itself.The pectin degrading enzyme can, for example, be a pectinestrerase,pectate lyase and/or a polygalacturonase.

Careful attention should be given to the emulsifier of choice. Lipidesterases are water soluble and therefore reside in the water componentof the emulsion, yet, their substrates reside in the oily portion of theemulsion. Presently preferred emulsifiers hence include lecithin,deoxycholate, gum Arabic (e.g., 0.5-2.0%), free fatty acid salts (e.g.,0.5-2.0%), bile derived emulsifiers and non-ionic detergents, such as,but not limited to, polyoxyethylensorbitane monolaurate (TWEEN-20).Preferably, the emulsifier employed is a non-ester emulsifier, as esteremulsifiers can adversely affect the reaction as competitive substratesor inhibitors of the esterase of choice. Suitable non-ester emulsifiersinclude, but are not limited to deoxycholate, gum Arabic (e.g.,0.5-2.0%) and free fatty acid salts (e.g., 0.5-2.0%).

While reducing the present invention to practice, it was uncovered thatthe effluent liquid phase remaining after removal of immobilizedesterase and solvent extraction of the carotenoid fraction following theenzymatic deesterification reaction of the present invention, containedconsiderable amounts of deoxycholate which, when dried, was reusable. Asdescribed in detail hereinbelow (see Example 6), by collection andlyophilization or oven drying of the aqueous effluent from theextraction of carotenoids following enzymatic deesterification,sufficient deoxycholate was recovered to provide efficientemulsification in additional enzymatic deesterification reactions (seeFIGS. 9 a-9 d hereinbelow). Reuse of the recovered, dried emulsifier iseffected by reconstitution with water.

As is shown in FIGS. 9 a-9 d, and FIG. 10 hereinbelow, the efficiency ofdeesterification of oleoresin carotenoids is not compromised by usingrecycled deoxycholate. Indeed, an unexpected synergistic result wasobserved, wherein the deesterification efficiency actually increasedwith repeated recycling of the deoxycholate (see, for example, FIG. 10).Without wishing to be limited by a single hypothesis, it is postulatedthat the increased efficiency of deesterifiecation using recovered,reconstituted recycled emulsifier as described herein, can be the resultof extraction of natural emulsifiers intrinsic to the startingcarotenoid source.

As regarding the reuse of immobilized lipase, the recovery and reuse ofrecycled emulsifiers, such as deoxycholate, for deesterification ofcarotenoids is of great advantage not only for the improved simplicityand reduction of costs that it affords, but also for the greatlysuperior purity of effluent wastes from the deesterification process,which is of crucial environmental and ecological concern, especiallywhere emulsifiers and detergents such as deoxycholate and Tween areinvolved (see, for example, the guidelines for handling of toxic andhazardous wastes in www.orcbs.msu.edu/newhazard/wastemanual).

Thus, according to the present invention there is provided a method ofincreasing a fraction of free carotenoids in a source of carotenoids inwhich at least some of the carotenoids are fatty acid esterifiedcarotenoids, the method comprising contacting the source of carotenoidswith an effective amount of an esterase and a recycled emulsifier underconditions effective in deesterifying the fatty acid esterifiedcarotenoids, thereby increasing the fraction of free carotenoids in thesource of carotenoids.

In one preferred embodiment, emulsifier is a non-ester emulsifier, suchas lecithin, deoxycholate and/or a non-ionic detergent. In yet anotherembodiment, the emulsifier is derived from bile, gum Arabic or salt offree fatty acids.

It will be appreciated, in the context of the present invention, thattraditional extraction processes for the manufacture of concentratedextracts (concentrated several fold as compared with the raw material)involve not only the use of various non-edible solvent systems, but alsoa large proportion of solvent in relation to the compounds of interest,which must be eliminated from the finished extracts. The last traces ofundesirable non-edible solvents are very difficult to separate from theconcentrated extract, limiting the potential use of the residual solidfor human consumption, and contributing to environmental contamination.Methods relying on high pressure and countercurrent extraction forproduction of highly concentrated carotenoid extracts have beendisclosed (see, for example, U.S. Pat. No. 5,773,075 to Todd, and5,789,647 to Heidlas et al), however, they require expensive equipment,involve undesirable temperatures, and are difficult to control.

As described hereinbelow, enzymatically deesterified red pepperoleoresin can be extracted with a non-polar solvent, such as hexane,chloroform, carbon tetrachloride, etc. While reducing the presentinvention to practice, it was surprisingly uncovered that extraction ofthe enzymatically deesterified carotenoids with a more polar solventunder mild alkaline conditions provided greatly enhanced efficiency,allowing for previously unattainable concentration and purity of thedeesterified carotenoid fraction.

As described in Example 7 hereinbelow, addition of base to make theextraction mixture mildly alkaline, and extraction with a polar solventsuch as ethyl acetate, chloroform, etc., results in unexpectedly highlyconcentrated deesterified red pepper oleoresin oil, comprisingexceedingly desirable proportions of deesterified carotenoids. As shownin HPLC chromatograms of FIGS. 13 a-13 b, ethyl acetate extraction (FIG.13 b) of the enzymatically deesterified red pepper oleoresin results ina concentrated product having a profile of deesterified carotenoidsequal to that produced with hexane extraction (FIG. 13 a). The volume ofthe polar solvent (ethyl acetate) required for extraction issignificantly less than that for the hexane (3-4 ml/5 mldeesterification reaction vs 7-8 ml/5 ml deesterification reaction).Comparison between the HPLC profiles from ethyl acetate extraction with(FIG. 13 b) and without (FIG. 13 a) mild alkaline pH adjustment of theextraction mixture reveals similarly efficient separation between thedeesterified carotenoids, and their mom- and di-ester forms.

However, comparison of the carotenoid composition (FIGS. 14 a and 14 b)reveals that extraction with a more polar solvent, under mild alkalineconditions, greatly enriches the carotenoid and Vitamin E content of theresulting deesterified red pepper carotenoid extract. Whereas ethylacetate extraction without pH adjustment actually reduces the carotenoidconcentration (FIG. 14 b), mild alkaline conditions improve theefficiency and purity of the extracted carotenoids, immensely (FIG. 14b). It will be noted that Vitamin E concentrations are enhanced evenmore significantly by mild alkaline ethyl acetate extraction.

Thus, according to the present invention there is provided a method ofincreasing a fraction of free carotenoids in a source of carotenoids inwhich at least some of the carotenoids are fatty acid esterifiedcarotenoids, the method comprising contacting the source of carotenoidswith an effective amount of an esterase under conditions effective indeesterifying the fatty acid esterified carotenoids, so as to produce asource of at least partially deesterified carotenoids, and extractingthe source of at least partially deesterified carotenoids with ethylacetate under alkaline conditions, thereby increasing the fraction offree carotenoids in the source of carotenoids.

In one preferred embodiment, the alkaline conditions are characterizedby pH from about 8.0 to about 10, most preferrably pH 9.5. Further, itwill be noted that polar solvents such as alcohols, chloroform, dimethylchloride, etc can be used in the mild alkaline extraction of theenzymatically deesterified carotenoids. Extraction and washing procedureis described in detail in the Materials and Experimental Proceduressection hereinbelow.

It will be noted that the aqueous fraction of the mild alkalineextraction described hereinabove, comprising the free fatty acidsreleased in the deesterification of the carotenoids, can be collected,acidified, and re-extracted with a solvent such as methyl acetate, andconcentrated by evaporation. The resulting concentrated source ofnatural free fatty acids from red pepper oleoresin can be used, forexample, as a food additive, a feed additive, or in cosmetic and/orpharmaceutical compositions.

The resultant source of carotenoids highly enriched in deesterifiedcarotenoids and Vitamin E is a superior source of antioxidants (see, forexample, FIG. 14). As shown in Example 8 hereinbelow, enzymaticallydeesterified red pepper oleoresin inhibited lipid oxidation withsignificantly greater efficiency than other well known carotenoidantioxidants lycopene and beta carotene (see FIGS. 16 a and 16 b).Further, the novel enzymatically deesterified carotenoid compositiondescribed herein exhibits superior antioxidant properties at a varietyof pH ranges, similar to those characteristic of different physiologicalconditions. Thus, at neutral pH, similar to blood and most tissueenvironments, deesterified carotenoid inhibited lipid oxidation 2-4 foldbetter than beta-carotene or lycopene, respectively, at 40 μMantioxidant (FIGS. 15 a and 16 a), and at highly acidic pH, such as indigestion in the stomach, 1-2 fold better, than lycopene orbeta-carotene, respectively (FIGS. 15 b and 16 b).

Thus, according to the present invention there is provided a compositionof matter comprising enzymatically deesterified red carotenoids, thecomposition of matter characterized by at least about 40 percent byweight capsanthin, at least about 15 percent by weight zeaxanthin andcapsolutein, at least about 2 percent by weight violaxanthin, at leastabout 1 percent by weight capsorubin, at least about 5 percent by weightbeta cryptoxanthin and at least about 3 percent by weight beta carotene,the composition characterized by antioxidant activity, as measured bylipid oxidation. The source of red carotenoids can be, as detailedabove, red pepper carotenoids, paprika oleoresin, red pepper or paprikapowder, etc.

As used herein in the specification and in the claims section below, theterm “inhibit” and its derivatives refers to suppress or restrain fromfree expression of activity.

As defined herein in the specification and in the claims section below,the term “about” refers to a value within 0.15 times greater or lesserthan the indicated value.

The composition of matter can comprise, or be depleted of, Vitamin E. Inone preferred embodiment, the composition of matter comprises at least 5mg per gram, most preferrably 20 mg per gram Vitamin E. Depletion ofVitamin E is effected by passage of the enzymatically deesterifiedcarotenoid preparation, following extraction, over a column of magnesiumsilicate, as described in Example 8 hereinbelow. Vitamin E is elutedwith hexane, while the enriched fraction of deesterified carotenoidsremains bound, and can be eluted with further washings with ethylacetate (See FIGS. 11 a and 11 b).

Thus, according to one aspect of the present invention there is provideda method of reducing a fraction of Vitamin E in a source of carotenoidsin which at least some of the carotenoids are fatty acid esterifiedcarotenoids, the method comprising contacting the source of carotenoidswith an effective amount of an esterase under conditions effective indeesterifying the fatty acid esterified carotenoids, so as to produce asource of at least partially deesterified carotenoids, andchromatographically extracting the fraction of Vitamin E away from saidsource of at least partially deesterified carotenoids, thereby reducingthe fraction of Vitamin E in the source of carotenoids.

Similarly, there is provided a food additive and/or a feed additivecomprising the composition of matter.

As defined herein in the specification and in the claims section below,the phrase “chromatographically extracting” is defined as separation ofa fraction or fractions from other components of a material as a resultof differential distribution of solutes as they flow around or over astationary liquid or solid phase. Well known examples of chromatographyinclude, but are not limited to liquid chromatography, gaschromatography, gas-liquid chromatography, affinity chromatography,paper chromatography, HPLC, etc. In one embodiment, chromatographicallyextracting the fraction comprises contacting the source of at leastpartially deesterified carotenoids with a magnesium silicate resin,washing with hexane, and eluting the at least partially deesterifiedcarotenoids with ethyl acetate. In another, more preferred embodiment,the magnesium silicate resin is Florisil (Supelco, Bellefonte, Pa.).

As described in detail in the Background section hereinabove, dietarycarotenoid intake has been inversely correlated with the incidence andseverity of a number of diseases and conditions, such as cancer,cardiovascular disease, age related degeneration, arthritis, etc., dueto the highly efficient antioxidant activity of carotenoids. Thus,carotenoids, which are widely distributed in nature, are natural dietarycomponents, and are well tolerated by human digestion, are in greatdemand as nutritional supplements, food additives, feed additives, andtherapeutic compositions (Hamilton Clin J Oncol Nurs 2001;5:181-2; Brownet al. Clin Excell Nurs Prac 998;2:10-22). The antioxidant propertiesand bioavailability of the red carotenoids in the novel enzymaticallydeesterified carotenoid composition of the present invention clearlysurpass those of previously available compositions. For example, apopular antioxidant supplement provides 5,712 mcg. beta carotene, 180mcg. alpha carotene, 36 mcg. zeaxanthin, 44 mcg. cryptoxanthin, 28 mcglutein., 100 i.u (150 mg) d-alpha tocopherol succinate (natural vitaminE) (taken twice to four times daily) (AntiOxidant Formula, PureEncapsulations, Sudbury, M A), the carotenoids being in native, mostlyesterified form. In sharp contrast, 1 gram of the enzymaticallydeesterified composition of the present invention comprises nearly 800times as much zeaxanthin and capsolutein and twice as much beta carotene(FIG. 14 b), having enhanced bioavailability in the deesterified form.Vitamin E content of 1 gram of the enzymatically deesterifiedcomposition alone is greater than the RDA of 15 mg (10 I.U.)/day(Dietary Ref Intakes for Vitamin C, Vitamin E, Selenium and Carotenoids,Institute of Medicine, 2000; 325-382, Nat'l Academies Press).

Thus, according to another aspect of the present invention there isprovided an article of manufacture comprising a packaging material andat least one antioxidant unit dosage, the antioxidant unit dosagecomprising a composition of matter comprising at least about 40 percentby weight capsanthin, at least about 15 percent by weight zeaxanthin andcapsolutein, at least about 2 percent by weight violaxanthin, at leastabout 1 percent by weight capsorubin, at least about 5 percent by weightbeta cryptoxanthin, at least about 3 percent by weight beta carotene andat least 20 mg per gram Vitamin E and a pharmaceutically acceptablecarrier in each single unit dosage. The packaging material comprises alabel or package insert indicating that the composition of matter is forincreasing antioxidant levels in a subject.

In one preferred embodiment, the composition of matter further comprisesa pharmaceutically acceptable excipient selected from the groupconsisting of carboxymethylcellulose, microcrystalline cellulose,starch, and modified starch.

In another preferred embodiment, the antioxidant unit dosage is designedfor oral administration. The antioxidant unit dosage can be selectedfrom the group consisting of a tablet, a caplet and a capsule. Thecomposition of matter can be in liquid dosage form.

The enzymatically deesterified carotenoids of the present invention canbe used to produce an antioxidant unit dosage. As used herein a“antioxidant unit dosage” refers to a preparation of one or more of theactive ingredients described herein, either carotenoids orphysiologically acceptable salts or prodrugs thereof, with otherchemical components such as traditional drugs, physiologically suitablecarriers and excipients. The purpose of an antioxidant unit dosage is tofacilitate administration of antioxidants to a cell or to an organism.Antioxidant unit dosage of the present invention may be manufactured byprocesses well known in the art, e.g., by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans.Hereinafter, the phrases “physiologically suitable carrier” and“pharmaceutically acceptable carrier” are interchangeably used and referto a an approved carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered conjugate.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the therapeutic is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of thecompound, preferably in purified form, together with a suitable amountof carrier so as to provide the form for proper administration to thepatient. The formulation should be suitable for the mode ofadministration.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate processes andadministration of the active ingredients. Examples, without limitation,of excipients include calcium carbonate, calcium phosphate, varioussugars and types of starch, cellulose derivatives, gelatin, vegetableoils and polyethylene glycols.

Further techniques for formulation and administration of activeingredients may be found in “Remington's Pharmaceutical Sciences,” MackPublishing Co., Easton, Pa., latest edition, which is incorporatedherein by reference as if fully set forth herein.

Various routes for the administration of active ingredients arepossible, and were previously described, for the purpose of the presentinvention. The topical carrier is one, which is generally suited fortopical active ingredients administration and includes any suchmaterials known in the art. The topical carrier is selected so as toprovide the composition in the desired form, e.g., as a liquid ornon-liquid carrier, lotion, cream, paste, gel, powder, ointment,solvent, liquid diluent, drops and the like, and may be comprised of amaterial of either naturally occurring or synthetic origin. It isessential, clearly, that the selected carrier does not adversely affectthe active agent or other components of the topical formulation, andwhich is stable with respect to all components of the topicalformulation. Preferred water-soluble ointment bases are prepared frompolyethylene glycols of varying molecular weight; again, reference maybe made to Remington: The Science and Practice of Pharmacy for furtherinformation.

Gel formulations are preferred for application to the scalp. As will beappreciated by those working in the field of topical active ingredientsformulation, gels are semisolid, suspension-type systems.

Various additives, known to those skilled in the art, may be included inthe topical formulations of the invention. For example, solvents may beused to solubilize certain active ingredients substances. Other optionaladditives include skin permeation enhancers, opacifiers, anti-oxidants,gelling agents, thickening agents, stabilizers, and the like.

Other agents may also be added, such as antimicrobial agents, antifungalagents, antibiotics and anti-inflammatory agents.

Other components, which may be present, include preservatives,stabilizers, surfactants, and the like.

The antioxidant unit dosage herein described may also comprise suitablesolid or gel phase carriers or excipients. Examples of such carriers orexcipients include, but are not limited to, calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin andpolymers such as polyethylene glycols.

Other suitable routes of administration may, for example, include oral,rectal, transmucosal, transdermal, intestinal or parenteral delivery,including intramuscular, subcutaneous and intramedullary injections aswell as intrathecal, direct intraventricular, intravenous,inrtaperitoneal, intranasal, or intraocular injections.

Compositions of matter for use in accordance with the present inventionthus may be formulated in conventional manner using one or morepharmaceutically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological salinebuffer. For transmucosal administration, penetrants are used in theformulation. Such penetrants are generally known in the art.

For oral administration, the active ingredients can be formulatedreadily by combining the active ingredients with pharmaceuticallyacceptable carriers well known in the art. Such carriers enable theactive ingredients of the invention to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for oral ingestion by a patient. Preparations for oral use can bemade using a solid excipient, optionally grinding the resulting mixture,and processing the mixture of granules, after adding suitableauxiliaries if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as,for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active ingredient doses.

Compositions which can be used orally, include push-fit capsules made ofgelatin as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. The push-fit capsules maycontain the active ingredients in admixture with filler such as lactose,binders such as starches, lubricants such as talc or magnesium stearateand, optionally, stabilizers. In soft capsules, the active ingredientsmay be dissolved or suspended in suitable liquids, such as fatty oils,liquid paraffin, or liquid polyethylene glycols. In addition,stabilizers may be added. All formulations for oral administrationshould be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the active ingredient and a suitable powderbase such as lactose or starch.

The active ingredients described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Suspensions of the active ingredients may be prepared as appropriateoily injection suspensions. Suitable lipophilic solvents or vehiclesinclude fatty oils such as sesame oil, or synthetic fatty acids esterssuch as ethyl oleate, triglycerides or liposomes. Aqueous injectionsuspensions may contain substances, which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the active ingredients to allowfor the preparation of highly concentrated solutions.

The compositions of matter of the invention can be formulated as neutralor salt forms. Pharmaceutically acceptable salts include those formedwith anions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The compositions of matter described herein described may also comprisesuitable solid of gel phase carriers or excipients. Examples of suchcarriers or excipients include, but are not limited to, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin and polymers such as polyethylene glycols.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any active ingredient used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromactivity assays in animals. For example, a dose can be formulated inanimal models to achieve a circulating concentration range that includesthe IC₅₀ as determined by activity assays. Such information can be usedto more accurately determine useful doses in humans. In general, dosageis from about 0.01 micrograms to about 100 g per kg of body weight, andmay be given once or more daily, weekly, monthly or yearly, or even onceevery 2 to 20 years.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures inexperimental animals, e.g., by determining the IC₅₀ and the LD₅₀ (lethaldose causing death in 50% of the tested animals) for a subject activeingredient. The data obtained from these activity assays and animalstudies can be used in formulating a range of dosage for use in human.

The dosage may vary depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain themodulating effects, termed the minimal effective concentration (MEC).The MEC will vary for each preparation, but can be estimated from invitro data; e.g., the concentration necessary to achieve 50-100%inhibition of lipid oxidation may be ascertained using the assaysdescribed herein. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. HPLC assays orbioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using the MEC value.Preparations should be administered using a regimen, which maintainsplasma levels above the MEC for 10-90% of the time, preferable between30-90% and most preferably 50-90%.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accompanied by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions of the invention formulated in a compatiblepharmaceutical carrier may also be prepared, placed in an appropriatecontainer, and labeled for treatment of an indicated condition.

The present invention further provides methods of (i) determining anefficiency of an esterase in increasing a fraction of free carotenoidsin a source of carotenoids in which at least some of the carotenoids arefatty acid esterified carotenoids; (ii) screening for esterasesefficient in increasing a fraction of free carotenoids in a source ofcarotenoids in which at least some of the carotenoids are fatty acidesterified carotenoids; (iii) optimizing reaction conditions forincreasing a fraction of free carotenoids in a source of carotenoids inwhich at least some of the carotenoids are fatty acid esterifiedcarotenoids, via an esterase; and (iv) increasing a fraction of freecarotenoids in a source of carotenoids in which at least some of thecarotenoids are fatty acid esterified carotenoids. The present inventionfurther provide a source of carotenoids having an increased fraction offree carotenoids, which can serve as a food and/or feed additive; and arich source from which one can extract to purification desiredcarotenoids.

The present invention offers a great advantage over processes forchemical deesterification of carotenoids. For example, alkalinetreatment of paprika affects to a great extent the properties of itsproteins and antioxidants such as vitamin C and E. It will beappreciated that during heating of paprika to high temperatures, asrequired in alkaline based deesterification of carotenoids, one or moreof the following adverse reactions takes place: (i) destruction ofessential amino acids; (ii) conversion of natural amino acids intoderivatives which are not metabolized; (iii) decrease of thedigestibility of proteins as a result of cross-linking; and, last, butnot least, generation of cytotoxic compounds. It will be appreciated inthis respect that due to the formation, at high pH values, of enolates,phenolic compounds, including vitamin E and most of the otherantioxidants are more rapidly oxidized, in a process that generates freeradicals which oxidize and destroy carotenoids (Belitz and Grosch W.Food Chemistry, Springer-Verlag, 1987).

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Materials and Experimental Procedures

Materials:

Paprika powder and oleoresin paprika were purchased fromTavlinei-Hanegev, Avshalom. Sodium phosphate, citric acid, TWEEN-20(polyoxyethylensorbitane monolaurate) and potassium hydroxide wereobtained from Merck (Darmstadt, Germany). Deoxycholic acid (sodium salt)BHT (Butylated hydroxy toluene), pancreatic lipase from porcine wereobtained from Sigma Chemical Co. (St. Louis, Mo.). The enzymes, lipase A“Amano 6”, lipase F-AP15 and lipase AY “Amano 30” (approved for humanconsumption) were from Amano, Pharmaceuticals Co. LTD (Nishiki, Japan).Candida rugosa lipase immobilized on porous acrylic beads was from Sigma(Cat #L1150, Sigma Chemical Co., St Louis Mo.). Pectinase/cellulase,Rohameut Max and protease (Coralase PN-L) were obtained from Rohm Enzymegmbh (Darmstadt, Germany). HPLC grade ethanol and hexane were fromBiolab (Israel) and HPLC acetone from Baker (Deventer, Holland). Ethylacetate was from BioLab (Israel).

High-Performance Liquid Chromatography (HPLC):

HPLC was conducted on a Shimadzu LC-10 AT equipped with SCL-10A Shimadzudiode array detector. Photodiode array measurements of spectralproperties from the individual peaks (from 260 to 540 μm) weredetermined at the upslope, apex and downslope. The column (Merck RP-18e3.4×250 mM, 5-μm particles) was used for HPLC separations. The peakswere detected at 450 and 474 nm. The mobile phase were acetone and H₂Owith a gradient as suggested by Minguez-Mosquera et al. 1993 (J. Agric.Food Chem. 41, 1616-1620).

Deesterification of Paprika Powder by Enzymes:

Paprika powder (500 mg) was suspended in 9.5 ml water in the presence ofCellulase-Pectinase (100 μl), Lipase (100 mg) and 0.2% deoxycholate (200mg) at pH 4.93. The suspension was shaken in a heated bath at 37° C. for24 hours. Carotenoids were extracted from the suspension by addition ofethanol (5 ml) and 5 ml of hexane. The extraction with hexane was donerepeatedly until no color could be observed in the extracts.

Deesterification of Paprika Oleoresin by Enzymes:

Paprika oleoresin (20 mg) was mixed with TWEEN-20 (200 μl) ordeoxycholate (100 mg) and 10 ml of H₂O. The emulsion has been shaken at37° C. for 24 hours. Extraction of carotenoids was performed by theaddition of 4 ml of ethanol and 5 ml of hexane. The extraction withhexane was done repeatedly until no color could be observed in theextracts. The combined hexane extracts were washed with water (25 ml)and dried over anhydrous sodium sulfate for HPLC determination of thecarotenoids.

Chemical Deesterification (Chemical Saponification):

Chemical deesterification was performed essentially as described inIttah et al., J. Agric. Food Chem. 1993, 41, 899-901.

Immobilized Lipase and Recycling:

Immobilized lipase from Candida rugosa (Sigma-Aldrich Corp., St Louis,Mo.). Following use in deesterification, the enzyme-bearing beads arerecovered by precipitation at 15 K RPM, and stored at 4° C. until reuse.

Recovery and Reconstitution of Deoxycholate:

Deoxycholate was recovered from the pooled aqueous phase of the ethylacetate extraction by freeze drying (Ilshin Laboratories) or oven dryingat 95° C. Briefly, following carotenoid extraction from the deesterifiedsolution, the aqueous phase is collected and dried to a powder. Therecovered deoxycholate was reconstituted with water before reuse inenzymatic deesterification. Reconstituted deoxycholate can be reused atleast 3 times.

Ethyl Acetate Extraction of Deesterified Carotenoids:

Following deesterification with lipase, the carotenoid fraction wasextracted by ethyl acetate, under alkaline pH. First, the lipasereaction mixture was made alkaline with NaOH, to pH 9.5 (with 0.1N NaOHsolution) to convert the free fatty acids products to water solublesalts. Ethyl acetate was then added at a ratio of 1 volume ethyl acetateto 5 volumes aqueous deesterification mixture, the phases mixed andallowed to separate. The aqueous phase was removed, the ethyl acetatephase reextracted four times with distilled water (water:ethylacetate=4:1 volume/volume), followed by drying by addition ofsuperfluous sodium sulfate. Ethyl acetate solvent was then evaporatedunder vacuum in a Rotovapor (Buchi), to produce paprika oleoresinenriched in free carotenoids. Free fatty acids in the aqueous phase arequantitatively recovered by acidification and extraction with anon-polar solvent such as methyl acetate.

Vitamin E Separation from Deesterified Paprika Oleoresin:

Vitamin E was removed chromatographically from paprika oleoresin bypassage though a magnesium-silicate absorbent column (Florisil, Supelco,Bellefonte, Pa.). A 30 cmX1cm column of Florisil was equilabrated withhexane, and the paprika oleoresin applied to the column (0.5 ml paprikaoil diluted with 0.5 ml ethanol at room temperature). The column wasthen flushed with hexane (flow rate 1 ml/min), until the carotenecarotenoids (yellow color) were eluted. The retained carotenoids,including the xanthophylls (capsanthin), were then eluted from thecolumn by extensive washing with ethyl acetate.

Stability of Deesterified Oleoresin in Water:

Stability of paprika oleoresin before and after enzymaticdeesterification was assessed spectrophotometrically in a water emulsioncontaining 0.15% or 0.03% Tween-20 (polyoxyethylene sorbitanmonolaurate, Amersham Biosciences Inc, Piscataway, N.J., USA), stored at4° C. Clarity, as color concentration, was measured in deesterified anduntreated oleoresin by visible range spectrophotometry, as absorbance(ΔOD) at 474 nm (red), after subtraction of background at 600-800 nm, insamples removed every 10 days for measurement of color concentration.

Lipid Oxidation Inhibition Assay:

Antioxidant activity was measured by inhibition of met-myoglobincatalyzed lipid oxidation, as changes in diene conjugated productsdetected at 234 nm. Carotenoids (enzymatically deesterified oleoresin,Vitamin E depleted enzymatically deesterified oleoresin, lycopene, andbeta-carotene) were added to concentrations of 10, 20 or 40 μM, asindicated herein, and results compared with oxidation of lipids incontrol reactions having no added inhibitors, under neutral (pH 7.0) andhighly acidic (pH 3.0) conditions.

Experimental Results EXAMPLE 1

Efficient Enzymatic Deesterification of Red Pepper Caritenoids

As described hereinabove, carotenoids, although of significantimportance to human nutritional and health, are poorly absorbed fromnatural sources due to the abundance of the mono-, di-, andtriesterified forms. Currently, chemical saponification is used fordeesterification, but is both costly and harsh to the resultingcarotenoids. In an effort to provide a gentle, more efficient method ofdeesterification of natural carotenoids, red pepper extracts werereacted with lipases and the resultant carotenoids analyzed by HPLC.

FIG. 1 demonstrates a chromatogram of HPLC fractionation of natural redpepper (paprika) carotenoids. The main carotenoid is capsanthin. Thefree unesterified capsanthin was eluted at about 9 min. Most of thecapsanthin is esterified as monoesters and diesters. The mono esterswere eluted in three major peaks after β-cryptoxanthin (14.33 min) andbefore β-carotene (18.9 min). The diesters were eluted as 7 major peaksbetween 22-26 min.

FIG. 2 demonstrates that following chemical saponification all the peaksof red pepper (paprika) diesters and monoesters carotenoids disappearedand the chromatogram contains mostly about 9 peaks of: (i) capsanthin(6.1 min); (ii) violaxanthin (7.36 min); (iii) capsanthin (8.89 min);(iv) cis-capsanthin (10.33); (v) capsolutein (10.83 min); (vi)Zeaxanthin (11.2 min); (vii) cis-Zeaxanthin (12.0 min); (viii)β-crypotxanthin (14.36 min); and (ix) β-carotene. The disadvantages ofchemical saponification are discussed hereinabove.

FIG. 3 demonstrates that incubation of red pepper (paprika) at 37° C.for 24 hours with a pectinase/cellulase [Rohament max (Rohm) 0.1% byweight], a protease [Corolase PN-L (Rohm) 0.1% by weight] that maceratethe pectins, proteins and cellulose, respectively, and a lipase (amano30, 0.1% by weight), results in deesterification of the monoesters anddiesters to the free carotenoids yielding a chromatogram which issimilar to the chromatogram obtained via chemical deesterification (FIG.2).

FIG. 4 demonstrates deesterification of paprika oleoresin followingincubation of the oleoresin in the presence of deoxycholate (4% byweight) and lipase (amano 30, 0.1% by weight) for 24 hours at 37° C.

Similar assays conducted with other lipases: pancreatic lipase, lipase A“Amano 6”, lipase F-AP15 gave far poorer results.

FIGS. 5 a-c demonstrate deesterification of paprika oleoresin followingincubation of the oleoresin in the presence of deoxycholate (2%, 3% or4% by weight, respectively) and lipase (amano 30, 0.1% by weight) for 48hours at 37° C. Note that similar carotenoid deesterification resultsare obtained with 3% and 4% deoxycholate, yet somewhat inferiorcarotenoid deesterification results are obtained with 2% deoxycholate.It will be appreciated that similar reaction optimizations can beperformed for all other reaction ingredients.

These results demonstrate that treatment with esterase, under theconditions described herein, can efficiently deesterify red peppercarotenoids. As described hereinabove, enzymatic deesterification ofpaprika carotenoids according to the methods described herein, prior toingestion of the red pepper carotenoids by human or animals, cansignificantly enhance the bioavailability of these compound from the gutto the plasma.

EXAMPLE 2

The Effect of CaCl₂ and NaCl on the Lipase Activity

The activity of lipase at pH 7.6 at 37.0° C. for 18 hours on thedeestrification of red-pepper carotenoids was measured in the presenceof CaCl₂ and NaCl. As shown in Table 1, below, the addition of CaCl₂ tothe reaction mixture, significantly increased lipase activity. TABLE 1Treatment % Deestrification Enzyme alone* 73 Enzyme + CaCl₂ 1.875 mM 78Enzyme + CaCl₂ 3.75 mM 82 Enzyme + CaCl₂ 7.5 mM 89*50 mg oleoresin, 400 mg deoxycholate, 250 mg lipase.In the presence of 150 mM NaCl without CaCl₂, the deestrification was of87%. Thus, the addition of a metal salt to the lipase reaction mixtureled to improved efficiency of lipase hydrolysis of the oleoresincarotenoids.

EXAMPLE 4 Extraction of Oleoresin from Fresh or Frozen Red-Pepper Fruits

Fresh or frozen red-pepper fruits (100 parts) were homogenized withdistilled water (40 parts) for 5 minutes to a juice. The juice wascentrifuged at 25,000 g for 20 minutes and the pellet, either directly,or frozen, was mixed with 2 parts of ethanol and 10 parts of ethylacetate. The elluent was homogenized for 1 minute. The solvents wereseparated from the dry material by centrifugation and evaporated at 45°C. under vacuum to receive red pepper oleoresin. The steps of the methodare schematically presented in the flow chart of FIG. 6.

EXAMPLE 5 Enzymatic Deesterification of Carotenoids from PaprikaOleoresin with Recycled Immobilized Lipase

Enzymatic deesterification in solution requires large amounts of lipaseenzyme, which is removed along with the aqueous phase during theextraction and washes of the deesterified carotenoid. In order todetermine the effect of immobilization and reuse of lipase enzymes onefficiency and quality of deesterification of red pepper carotenoids,paprika oleoresin was deesterified with freshly prepared, and recycledmatrix-immobilized C. rugosa lipase.

As is shown in the HPLC chromatogram of the carotenoids followingdeesterification and extraction, (FIG. 7 a) matrix-immobilized lipaseequivalent to 100 mg enzyme is equally efficient in deesterifying redpepper carotenoids in paprika oeloresin as freshly-prepared, unboundlipase in suspension (compared with FIG. 5 a-5 c).

Immobilized lipase beads may be recycled by precipitation, and reused.However, reuse of immobilized enzymes often incurs deterioration oralteration of activity. Thus, the immobilized lipase was tested forefficiency of deesterification of carotenoids in paprika oleoresinfollowing 1 and 2 cycles of recovery. 20 mg of paprika oleoresin indeoxycholate and water emulsion was incubated with shaking for 24 hourswith the lipase beads, followed by removal of the beads, extraction andanalysis of the carotenoids. The identity of the. HPLC chromatogramsusing freshly-prepared immobilized lipase (FIG. 7 a), once recycled(FIG. 7 b) and even twice recycled (FIG. 7 c) enzyme indicate the neartotal retention of deesterification activity of recycled, immobilizedlipase. FIG. 8 compares the efficiency of deesterification with fresh,once-, and twice-recycled immobilized lipase, quantifying the efficientreuse of the immobilized enzyme with paprika oleoresin. Loss of enzymeactivity was less than 6% after 3 cycles of recovery and reuse. Thus,the immobilized lipase can be recycled numerous times withoutsignificant loss or alteration of activity.

EXAMPLE 6 Enzymatic Deesterification of Carotenoids from PaprikaOleoresin with Recycled Deoxycholate

Enzymatic deesterification in solution requires large amounts of theemulsifier, deoxycholate, which is removed along with the aqueous phaseduring the extraction and washes of the deesterified carotenoid.Recovery and reuse of the deoxycholate could be a significantimprovement, reducing the amounts of deoxycholate in waste, anddecreasing costs of the deesterification process. In order to determinethe effect of recovery and reuse of deoxycholate on efficiency andquality of deesterification of red pepper carotenoids, paprika oleoresinwas deesterified in the presence of freshly prepared, and dried,recycled deoxycholate.

As is shown in the HPLC chromatograms of the carotenoids followingdeesterification and extraction, (FIG. 9 b to 9 d), recovered andreconstituted deoxycholate is equally efficient in deesterifying redpepper carotenoids in paprika oeloresin as freshly-prepared deoxycholate(compared with FIG. 9 a). Deoxycholate was recovered from the aqueousphase following extraction and wash by either drying to a powder in anoven at 95° C. or freeze-drying (Ilshin Laboratories). Reconstitutionwas with water.

While reducing the present invention to practice, it was surprisinglydiscovered that recovery and recycling of deoxycholate resulted inincreased efficiency of enzymatic deesterification of red peppercarotenoids, compared with freshly prepared deoxycholate. FIG. 10compares the efficiency of deesterification with fresh (100%), once-,twice- and thrice-recycled deoxycholate, quantifying the efficient reuseof the emulsifier with paprika oleoresin. Clearly, the recovery andreuse of the deoxycholate enhances the enzymatic deesterification.

Without wishing to be limited by single hypothesis, the enhancedefficiency with recycling of deoxycholate may be due to the repeatedextraction of natural emulsifiers present in the paprika oleoresin, andtheir cumulative addition to the deesterification reaction.

EXAMPLE 7 Mild Alkaline Extraction of Enzymatically DeesterifiedCarotenoids with Ethyl-Acetate and Production of a Novel, Vitamin EEnriched Carotenoid Composition

Extraction of the enzymatically deesterified paprika oleoresincarotenoids has traditionally involved nonpolar solvents such as hexane.However, without wishing to be limited by one hypothesis, it is possiblethat since deeseterification of the carotenoids renders them more polarthan the esterified starting material, extraction with highly non-polarsolvents incurs a loss of carotenoid material, increased time ofextraction, and need for large volumes. Equally important, hexane andother highly non-polar solvents have been designated “Solvents ThatShould Be Limited” by the FDA, while more polar solvents such as ethylacetate are considered solvents with “Low Toxic Potential” (see VICHGL18, “Impurities Solvents, June 2000, FDA, U.S. Department of Healthand Human Services). In order to test the effects of extraction ofenzymatically deesterified carotenoids with a more polar solvent, mildalkaline extraction of deesterified red pepper carotenoids with ethylacetate was performed.

The separation by ethyl acetate was found to be simple, fast andrequired 4 times less solvent than separation by hexane (2-3 ml ethylacetate/5 ml deesterification reaction, compared to 7-8 ml hexane/5 mldeesterification reaction). Further efficiency of extraction waseffected by performing the extraction in a basic environment. In orderto increase the concentration of the carotenoids in the organic phase,free fatty acids from the deesterification were titered to the salt formby increasing the pH to 9.5, using NaOH. Ethyl acetate (about 1 volumesolvent to about volumes of aqueous solution) was added, the carotenoidsextracted, and the extracted oeloresin washed at least 3× with water(1:1 v/v). In the next stage, sodium sulfate was added to ethyl acetateto remove water from the ethyl acetate and dry it. The extractedcarotenoids were concentrated by evaporation of ethyl acetate by avacuum evaporator (Büchi) to a paprika oleoresin with free carotenoids.After mild alkaline ethyl acetate extraction, and concentration, theenzymatically deeseterified paprika oleoresin contains about 3 foldgreater carotenoids in the oil than the paprika oleoresin beforeenzymatic deesterification (from 74 mg carotenoids/1 g oil to 210 mg/1 goil).

FIGS. 13 a and 13 b show the qualitative and quantitative advantage ofethyl acetate extraction. Whereas the HPLC chromatograms of the twoenzymatically deesterified paprika oleoresin carotenoin preparationsindicate an identical profile of highly deesterified carotenoids (fromabout 6.8 minutes to about 11.6 minutes), the concentrations ofcarotenoids (162 mg carotenoids/1 g deesterified extract with ethylacetate, compared with 12.5 mg carotenoids/g with hexane) is far greaterwith the mild alkaline ethyl acetate extraction.

FIGS. 13 c and 13 d show the importance of mild alkalinization forefficient ethyl acetate extraction of enzymatically deesterified redpepper carotenoids. Whereas the HPLC chromatograms of the twoenzymatically deesterified paprika oleoresin carotenoin preparationsindicate an identical profile of highly deesterified carotenoids (fromabout 6.8 minutes to about 11.6 minutes), the concentrations ofcarotenoids (210 mg carotenoids/1 g deesterified extract with mild1alkaline ethyl acetate extraction, compared with 74.9 mg carotenoids/gwith ethyl acetate extraction without pH adjustment) is far greater withthe mild alkaline ethyl acetate extraction.

Paprika oleoresin is rich in Vitamin E, an extremely importantantioxidant and nutritional supplement. While reducing the presentinvention to practice, it was surprisingly discovered that the mildalkaline ethyl acetate extraction of enzymatically deesterified redpepper carotenoids produced a novel red pepper oeloresin compositionhighly enriched in capsanthin, zeaxanthin, capsolutein and Vitamin E.FIGS. 14 a and 14 b show the previously unattainable enrichment of theseimportant carotenoids, as well as that of Vitamin E, by mild alkalineethyl acetate extraction after enzymatic deesterification.

Free fatty acids removed from the enzymatically deesterified carotenoidsby the mild alkaline ethyl acetate extraction remain in the aqueousphase due to their conversion to salts in the elevated pH. Collectionand re-acidificaction of this fraction, after separation from theorganic phase, can render the free fatty acids extractable with asolvent such as methyl acetate, which, after a number of re-extractions,can be evaporated as described hereinabove, to provide a compositioncomprising concentrated, purified natural red pepper free fatty acidssuitable for nutritional, food or feed supplementation.

EXAMPLE 8 Antioxidant Activity of Enzymatically Deesterified Red PepperCarotenoids: Inhibition of Lipid Oxidation

As described in the Background section hereinabove, carotenoids areimportant as food additives and nutritional supplements for theirantioxidant properties. Oxidative modification of lipids andlipoproteins, which is thought to be a key step in the pathogenesis ofcancer, cardiovascular disease and other conditions, is protected byantioxidants. However, certain of the carotenoids shows less consistentprotective ability (Gaziano et al., 1995; Reaven et al., 1994). Mixturesof carotenoids have been found to be more effective as antioxidants thanany single carotenoid (Stahl et al., 1998). Moreover, it has beenreported that carotenoids enhance vitamin E antioxidant efficiency (Bohmet al., 1997; Fuhrman et al., 1997; Fuhrman and Aviram, 1999). In orderto assess the antioxidant character of enzymatically deesterified redpepper carotenoids, the effect of enzymatically deesterified carotenoidson lipid oxidation in a carotene-linoleic acid assay was determined.

As described hereinabove, Vitamin E (mostly mixed tocopherols) is asignificant component of natural sources of carotenoids, such as redpepper oleoresin. Vitamin E is also extracted and concentrated alongwith the carotenoid fraction, as demonstrated in FIG. 14. As animportant antioxidant micronutrient, the presence of Vitamin E in thecarotenoid composition is advantageous. However, in order to accuratelyassess the antioxidant properties of enzymatically deesterified redpepper carotenoids, a method of depleting Vitamin E without altering theintrinsic properties of the enzymatically deesterified carotenoids isrequired.

While reducing the present invention to practice, it was found thatpassage of the concentrated, enzymatically deesterified red pepperoleoresin over a column of magnesium silicate (Florisil, Supelco,Bellefonte, Pa., USA) equilibrated with hexane allowed separation ofyellow and red (xanthophyll) carotenoids. Elution of the retained redcarotenoids with ethyl acetate yielded quantitative recovery of redcarotenoids, depleted of Vitamin E by more than 40-fold (compare HPLC ofVitamin E in FIG. 11 a and FIG. 11 b). Thus, the antioxidant effects ofthe enzymatically deesterified red pepper oleoresin carotenoids couldthen be accurately asessed.

When compared to lycopene (x's, FIGS. 15 a and b) and beta carotene(stars, FIGS. 15 a and 15 b), the enzymatically deesterified red peppercarotenoids showed superior inhibition of lipid oxidation (FIGS. 15 aand 15 b, closed squares and closed triangles), at both neutral pH (asis found in the blood, for example) (FIG. 15 a), and strongly acidic pH(as is found in the stomach, for example) (FIG. 15 b). Both Vitamin Econtaining (closed squares, FIGS. 15 a and 15 b), and Vitamin E-depleteddeesterified red pepper carotenoids (closed triangles, FIGS. 15 a and 15b) showed similar, superior antioxidant properties irrespective of pH.

The bar graphs of the effect of carotenoid concentration on theantioxidant character of enzymatically deesterified carotenoids in FIGS.16 a and 16 b clearly demonstrate that at all concentrations and at bothneutral and alkaline pH, both Vitamin E containing (“Capsivit”, FIGS. 16a and 16 b), and Vitamin E-depleted deesterified red pepper carotenoids(“saponified carotenoids”, FIGS. 16 a and 16 b) showed similar, superiorantioxidant properties.

EXAMPLE 9 Stability of Enzymatically Deesterified Red Pepper Carotenoids

In order to determine the stability of enzymatically deesterified redpepper carotenoids, the color concentrations of emulsions prepared frompaprika oleoresin before and after enzymatic deesterification and mildalkaline ethyl acetate extraction were compared over a 30 day storageperiod. The color concentration was determined in the presence of twoconcentrations of non-ionic, non-denaturing detergent (Tween 20) toassess the dispersal of the carotenoids in the water.

The Table in FIG. 12 shows the superior stability of the enzymaticallydeesterified paprika oleoresin in water, upon preparation (Time 0), andafter 10 days storage (After 10 days). Comparing the absorbance valuesat 474 nm, the enzymatically deesterified oleoresin (1 mg/100 ml water)clearly retained greater than 97% of the color concentration at 0.15%Tween-20 (1.402 vs 1.377) and at 0.03% Tween 20 (1.352 vs 1.326), withonly a small difference between the color concentration of the emulsionin higher (0.15%) and lower (0.03%) Tween concentrations. In sharpcontrast, the untreated, esterified paprika oleoresin (1 mg/100 mlwater) retained only around 70% of initial color concentration after 10days storage (0.912 vs 0.662) in lower Tween (0.03%) concentration, andonly 95% (1.220 vs 1.159) at the higher Tween (0.15%) concentration.

Further, it was observed that upon standing, the emulsion prepared fromthe untreated, esterified paprika oleoresin, but not the enzymaticallydeesterified paprika oleoresin, developed a ring of oil at thebottleneck of it's container, along with the loss of colorconcentration. Without wishing to be limited to a single hypothesis, theloss of color concentration is most likely caused by hydrophobicinteractions and accumulation of the untreated, esterified carotenoidsin the oily ring.

Thus, the enzymatically deesterified, mild alkaline ethyl acetatepaprika oleoresin has superior storage and dispersive properties in anaqueous solution, even at low detergent concentations.

Taken together, these results show that: i) red carotenoids, such asthose found in red pepper fruits, can be efficiently and quantitativelydeesterified by gentle enzymatic hydrolysis, using the novel conditionsdescribed hereinabove, to produce a deesterified red carotenoidcomposition equal if not superior to that produced by chemicalsaponification; ii) immobilized enzymes and recovered, recycledemulsifiers, such as deoxycholate, can be used and reused in thedeesterification process without significant loss of quality or quantityof the deesterified carotenoid product; iii) novel, efficient extractionmethods using ethyl acetate and mild alkaline conditions produce apreviously unattainable composition comprising high concentrations ofenzymatically deesterified red carotenoids and Vitamin E, as well aspurified, isolated carotenoid free fatty acids; Vitamin E can bequantitatively depleted from the enzymatically deesterified red peppercarotenoids chromatography; and v) enzymatically deesterified red peppercarotenoids have superior antioxidant characteristics, with, or withoutthe Vitamin E component enriched by enzymatic deesterification andextraction described hereinabove.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

REFERENCES CITED Additional References are Cited in the Text

-   Akhtar P., Gray I J., Thomas H C., Garling D L. And Booren Am.    Dietary pigmentation and carotenoids in rainbow trout muscle and    liver tissue. J. Food Chem. 1999, 64, 234-239.-   Aviram M. Review of human studies on oxidative damage and    antioxidant protection related to cardiovascular diseases. Free    Radic. Res. 1999, (in press).-   Aviram M. Paraoxonase protects lipoproteins foam oxidation and    attenuates atherosclerosis. Cardiovas. Res. 1999 (in press).-   Aviram M, Maro I, Keidar S, Hayck T et al., Lesioned low-density    lipoprotein in atheroscelrotic aplipoprotein E-deficient transgenic    mice and human is oxidized and aggregated. Biochem. Biophys. Res.    Commun. 1995, 16, 501-513.-   Aviram M. Oxidized low density lipoproteins (OX-LDL) interaction    with macrophages in atherosclerosis and the antiatherogenicity of    antioxidants. Europ. J. Clin. Chem. Clin Biochem. 1996, 34, 599-608.-   Birchall M A, Schock E, Harmon B V, Gobe G. Apoptosis, mitosis, PCNA    and bcl-2 in normal, leukoplakic and malignant epithelia of the    human oral cavity: prospective, in vivo study. Oral Oncol    1997,33,419-425-   Block G, Patterson B, Subar A. Fruit, vegetables and cancer    prevention. A review of the epidemiological evidance. Nutr. Cancer,    1992, 18, 3-4.-   Bohm F, Edge R, Land J E, McGravey D J, Triscott J G. Carotenoids    enhance vitamin E antioxidant efficiency. J. Am. Chem. Soc. 1997,    119, 621-622.-   Bosland P W. Breeding for quality in Capsicum. Capsicum Eggplan    News1. 1993. 12, 25-28.-   Bras A, Sanches R, Cristovao L, et al. Oxidative stress in familial    adenomatous polyposis. Eur J Cancer Prev 1999, 8, 305-310.-   Britton G. In Natural Food Clorants (Hendry G A Fand Houghton J. D.    eds) Blockie Academic Professional, London, 1996, p. 197.-   Bundgaard T, Wildt J, Frydenberg M, Elbrond O, Nielsen J E.    Case-control study of squamous cell cancer of the oral cavity in    Denmark. Crit Rev Oral Biol Med 1995, 6, 5-17.-   Burton G W, Ingold K U. β-carotene: An unusual type of lipid    antioxidant. Science, 1984, 224, 569-573.-   Collins A R, Gedik C M, Olmedilla B, Southon S, Bellizi M. Oxidative    DNA damage measured in human lymphocytes: large differences between    sexes and between countries, and correlation with mortality rates.    FASEB J 1998, 12, 1397-400.-   Cowan C G, Calwell E I L, Young I S, McKillop D J, Lamey P-J:    Antioxidant status of oral mucosal tissue and plasma levels in    smokers and non-smokers. J Oral Path Med 1999, 28, 360-363.-   Dammer R, Neiderdellman H, Friesenecker J, Fleisschmann H, Hermann    J, Kreft M. Withdrawal therapy of patients with alcoholism and    nicotine dependence with carcinomas in the area of the head a neck.    Luxury or necessity? Carcinogenesis 1998, 19, 509-514.-   De Stefani E, Boffetta P, Oreggia F, Mendilaharsu M,    Deneo-Pellegrini H. Smoking patterns and cancer of the oral cavity    and pharynx: a case control study in Urugay. Indian J Cancer 1998,    35, 65-72.-   Dugas T R, Morel D W, Harrison E H. Dietary supplementation with    β-carotene, but not with lycopene, inhibits endothelial all-mediated    oxidation of low-density lipoprotein. Free Rad. Biol. Med. 1999, 26,    1238-1244.-   Esterbauer H, Dieber-Rotheneder M, Striegl G, Waeg G. Role of    vitamin E in preventing the oxidation of low-density lipoproteins.    Am. J. Chim. Nutr. 1991, 53, 3145-3215.-   Esterbaur H, Cheseman K H. Determination of aldehydic lypid    peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods    Enzymol 186, 407-421.-   Everett J A, Dennis M F, Patel K B, Maddix S, Kunder S C, Wilson    R L. Scavenging of nitrogen dioxide, thiyl and sulfonyl free    radicals by the nutritional antioxidant β-carotene. J. Biol. Chem.    1996, 271, 2988-2991.-   Frankel E N, Kanner J, German J B, Kinsella E J. Inhibition of    oxidation of human low-density lipoprotein with phenolic substances    in red-wine Lancet 1993, 341, 454-457.-   Fuhrman B, Elis A. and Aviram M. Antiathrogenic effects of lycopene    and β-carotene: inhibition of LDL oxidation, and suppression of    cellular cholesterol synthesis. Natural Antioxidants and    Anticarcinogenesis in Nutrition Health and Disease. Eds.    Kumpulainen J. T. and Salonen J. T. Society of Chemistry, Cambridge,    U.K. 1999, pp. 226-230.-   Fuhrman B, Lavy A, and Aviram M. Consumption of red wine with meals    reduces the susceptibility of human plasma and LDL to undergo lipid    peroxidation. Am. J. Clin. Nutr. 1995, 61, 549-554.-   Gaziano J M, Hatta A, Flynn M, Johnson E J et al., N I, Ridker P M,    Henekens C H, Frei B. Supplementation with beta-carotene in vivo and    in vitro does not inhibit low density lipoprotein oxidation.    Atherosclerosis 1995, 112, 187-195.-   Gerster H. The potential role of lycopene for human health. J. Am.    Cell. Nutr. 1997, 16, 109-126.-   Goldsworthy T L, Conolly R B, Fransson-Steen R. Apoptosis and cancer    risk assessment. Mutat Res 1996, 365, 71-90.-   Goodwin T W: “The Biochemistry of the Carotenoids” Vol. 1: “Plants”.    New York, Chapman and Hall, 1980, p. 203.-   Gravis G, Pech-Gourgh F, Viens P, Alzieu C, Camerlo J, Oziel-Taieb    S, Jausseran M, Maraninchi D. Phase II study of a combination of    low-dose 13-cis-retinoic acid and interferon-alpha in patients with    advanced head and neck squamous cell carcinoma. Anticancer Drugs    1999, 10, 369-374.-   Halliwell B. Cellular stress and protection mechanism. Biochem. Soc.    Trans. 1996, 24, 1023-1027.-   Hart A k, Karakala D W, Pitman K T, Adams J F. Oral and    oropharyngeal squamous cell carcinoma in young adults: a report on    13 cases and review of the literature. Carcinogenesis 1999, 20    743-748.-   Hennekens C H, Buring J E, Manson J E, Stampfer M et al. Lack of    effect of long-term supplementation with beta-carotene on the    incidence of malignant neoplams and cardiovascular disease. N.    Engl. J. Med. 1996, 334, 1145-1149.-   Hertog M G L, Feskens E J M, Hollman P C H, Katan M B, et al.    Dietary antioxidants flavonoids and risk of coronary heart disease:    The Zutphen Eldery Study Lancet 1993, 342, 1007-1011.-   Hirayama O, Nakamura K, Hamda S, Kobayasi Y. Singlet oxygen    quenching ability of naturally occurring carotenoids. Lipid, 1994,    29, 149-151.-   Hong W K, Lippman S M, Itri L M, et al. Prevention of second primary    tumors with isotretinoin in squamous-cell carcinoma of the head and    neck. N Engl J Med 1990; 323:795-801-   Ilyas M, Straub J, Tomlinson I P, Bodmer W F. Genetic pathways in    colorectal and other cancers. Eur J Cancer 1999, 35, 335-351.-   Iribarren C, Folsom A R, Jacobs D R Jr et al. Association of serum    vitamin levels, LDL susceptibility to oxidation and autoantibodies    against MDA-LDL with carotid atherosclerosis. Arterioscler. Tromb.    Vase Biol. 1997, 17, 1171-1177.-   Jain C K, Agarwal S, Venketeshwer R. The effect of dietary lycopene    on bioavailability, tissue distribution, in vivo antioxidant    properties and colonic preneoplasia in rats. Nutr Res 1999, 191,    383-391.-   Kanner J, and Kinsella, J E, Lipid deterioration: β-carotene    destruction and oxygen evolution in a system containing    lactoperoxidase, hydrogen peroxide and halides. Lipids. 1983, 18,    198.-   Kanner J, Frankel E, Granit R, German B, and Kinsella E, Natural    antioxidants in grapes and wines. J. Agric. Food Chem. 1994, 42,    64-69.-   Kennedy T A, Liebler D C. Peroxyl radical scavenging by β-carotene    in lipid bilayers. J. Biol. Chem. 1992, 267, 4658-4663.-   Khachik F. Beecher G R, Smith J C. Lutein, lycopene and their    oxidative metabolites in chemoprevention of cancer. J. Cell Biochem.    1995, 22, 236-246.-   Kim D J, Takasuka N, Kim J M, Sekine K, Ota T, Asamoto M, Murakoshi    M, Nishino H, Nir Z, Tsuda H (1997) Chemoprevention by lycopene of    mouse lung neoplasia after combined initiation treatment with DEN,    MNU and DMH. Cancer Lett 120,15-22.-   Kim J M, Araki S, Kim D J, Park C B, Takasuka N, Baba-Toriyama H,    Ota T, Nir Z, Khachik F, Shimidzu N, Tanaka Y, Osawa T, Uraji T,    Murakoshi M, Nishino H, Tsuda H (1998) Chemopreventive effects of    carotenoids and curcumins on mouse colon carcinogenesis after    1,2-dimethylhydrazine initiation. Carcinogenesis 19,81-85.-   Knekt P, Jarvinen R, Reunaneu A, Maatek. Flavonoid intake and    coronary mortality in Finland: a cohort study. Brit. Med. J. 1996,    312, 478-481.-   Knudsen K E, Weber E, Arden K C, Cavenee W K, Feramisco J R, Knudsen    E S. The retinoblastoma tumor suppressor inhibits cellular    proliferation through two distinct mechanisms inhibition of cell    cycle progression and induction of cell death. Oncogene 1999, 16,    5239-5245.-   Kohlmeier L, Hossting S B. Epidemiologic evidence of a role of    carotenoids in cardiovascular disese prevention. Am. J.Clin. Nutr.    1995, 62, 137s-146s.-   Kohlmeier L, Kark J D, Gomez-Grania E, et al. Lycopene and    mycoradial infraction risk in the EURAMIC study. Am. J. Epidemiol.    1997, 146, 618-622.-   Kondo K, Matsumoto A k, Kusata H, Tenahashi H, Koda H, et al.    Inhibition of oxidation of low-density lipoprotein with red-wine.    Lancet, 344, 1152-1152.-   Kristenson M, Zieden B, Kuinkiene S, et al. Antioxidant state and    mortality from coronary heart disease in Lithuanian and Swedish men.    B.M.J. 1997, 314, 629-632.-   Lapidot, T. Harel, S. Akiri, B. Granit, R. and Kanner, J.    PH-Dependent forms of red wine anthocyanins as antioxidants. J.    Agric. Food Chem. 1999, 47, 67-70.-   Lapidot, T. Harel, S. Granit, R. Kanner, J. Anthocyanins in red    wines: Antioxidant activity and bioavailability in human. In Natural    1999, 151-161.-   Lee C M. Borleau A. Boileau T WM, Williams A W. Et al. Review of    animal models in carotenoid research. J. Nutr. 1999, 129, 2271-2277.-   Lee I M. Cook N R. Monson J E. Buring J E. Hennekens C H. B-carotene    supplementation and incidence of cancer and cardiovascular disease:    the women's study. J. Natl. Cancer Inst. 1999, 91, 2102-2102.-   Lefebvre V, Kunz M, Camara B. and Palloix A. The    capsanthin-capsorubin synthase gene: candidate for the y locus    controlling the red fruit color in pepper. Plant Molec. Biol. 1998.    36, 785-789.-   Levy A, Harel S, Palevich D, Akiri B, Menagem E, and Kanner J.    Carotenoid pigments and β-carotene in paprika fruit (Capsicum spp.)    with different genotypes. J. Agric. Food Chem. 1995. 43, 362-367.-   Levy A, Levy Talia, S, Elikin Y, Menagem E, Barzilai M, and    Kanner J. Carotenoid and vitamin C and E contents in isogenic    chlorophyll and color mutants of paprika (Capsicum annuum L.). Proc.    Xth. Eucarpia Meeting on Genetics and Breeding of Capsicum and    Eggplant. 1998, 257-260.-   Levy J. Bosin E, Feldman B, Giat Y et al. Lycopene is a more potent    inhibitor of human cancer cell proliferation than lither α-carotene    of β-carotene. Nutr. Cancer 1995, 24, 257-267.-   Lin Y, Burri B J, Neidlinger T R, Muller H G, Ducker S R, Cliford    A J. Estimating the concentration of beta-carotene required for    maximal protection of low-density lipoprotein in women. Am. J. Clin.    Nutr. 1998, 67, 837-845.-   Mao L. Leukoplakia: Molecular understanding of pre-malignant lesions    and implications for clinical management. Mol Med Today 1997, 3,    442-448-   Mathews Roth M M, Welankiwar S, Sehgal P K, Lausen N L G et al.    Distribution of (¹⁴C) lycopene in rats and monkey. J. Nutr. 1990,    120, 1205-1213.-   Matsufuji H, Nakamura H, Chino M and Takeda M. Antioxidant activity    of capsanthin and the fatty acid estess in paprika (Capsicum    annuum). J. Agric. Food Chem. 1998, 46-49.-   Mayne S T, Beta-carotene, carotenoids and disease prevention in    human, FASEB J. 1996, 10, 690-699.-   Murakoshi M, Nishino H, Satomi Y, Takayasu J et al. Potent    preventive action of α-carotene against carcinogenesis spontaneous    liver carcinogenesis in mice are suppressed more effectively by    β-carotene. Cancer Res. 1992, 52, 6583-6587.-   Narisawa T, Fukaura Y, Hasebe M, Ito M, Aizawa R, Murakoshi M,    Uemura S, Khachik F, Nishino H (1996) Inhibitory effects of natural    carotenoids, alpha-carotene, beta-carotene, lycopene and lutein, on    colonic aberrant crypt foci formation in rats. Cancer Lett    107,137-142.-   Ojima F, Sakamoto H, Ishiguro Y, Ferao J. Consumption of carotenoids    in photosensitized oxidation of human plasma and low-density    lipoprotein. Free Rad. Biol. Med. 1993, 15, 377-384.-   Olson J A. Carotenoids, In: Modem Nutrition in Health and Disease    (Shils M E, Olson J A, Shike M. & Ross A C eds) Williams and    Wilkins, Baltimore, Md. 1999, p. 525.-   Oshima S, Sakamoto H, Ishiguro Y and Terao J. Accumulation and    clearnce of capsanthin in blood plasma after the ingestion of    paprika juice in men. J. Nut. 1997, 127, 1475-1479.-   Pappalardo G, Maiani G, Mobarhan S, Guadalaxara A, Azzini E,    Raguzzini A, Salucci M, Serafini M, Trifero M, Illomei G,    Ferro-Luzzi A (1997) Plasma (carotenoids, retinol, alpha-tocopherol)    and tissue (carotenoids) levels after supplementation with    beta-carotene in subjects with precancerous and cancerous lesions of    sigmoid colon. Eur J Clin Nutr 51, 661-666.-   Poulos J. Pepper breeding (Capsicum spp.): achievements, challenges    and possibilities. Plant Breeding Absr. 1994, 64, 143-146.-   Rao A V and Agarwal S. Role of lycopene as antioxidant carotenoid in    the prevention of chronic disease: A review. Nutr. Res. 1999, 19,    305-323.-   Reaven P D, Ferguson E, Navab M, Powell F L. Susceptibility of human    LDL to oxidative modification. Effects of variations in    beta-carotene concentration and oxygen tension. Alterioscler. Troub.    1994, 14, 1162-1169.-   Romanchik J E, Morel D W, Horrison E H. Distribution of carotenoids    and alpha-tocopherol among lipoproteins do not change when human    plasma is incubated in vitro. J. Nutr. 1995, 88, 1646-1650.-   Ross R. The pathogenesis of atherosclerosis: a perspective for    the 1990. Nature, 1993, 362, 801-809.-   Schildt E B, Eriksson M, Hardell M, Magnuson A. Oral snuff, smoking    habits and alcohol consumption in relation to oral cancer in a    Swedish case-control study. Int J Cancer 1998, 77, 333-336-   Schoelch M L, Le Q T, Silverman S Jr, McMillan A, Dekker N P, Fu K    K, Ziober B L, Regezi J A. Apoptosis-associated proteins and the    development of oral squamous cell carcinoma. Oral Oncol 1999, 35,    77-85.-   Schroeder W A and Johnson E A. Singlet oxygen and peroxyl radical    regulate carotenoid biosynthesis in Phaffia Rhodozyma. J. Biol.    Chem. 1995, 270, 18374-18379.-   Schwartz J L and Shklar G. Retinoid and carotenoid angiogenesis: a    possible explanation for enhanced oral carcinogenesis. Nutr Cancer    1997, 27, 192-99.

Schwartz J L and Shklar G. The selective cytotoxic effect of carotenoidsand alpha tocopherol on human cancer cell lines in vitro. J Oral MaxillSurg 1992, 50, 367-373.

-   Schwartz J L Tanaka J, Khandekar V, Herman T S, Teicher B. Beta    carotene and/or vitamin E as modulators of alkakylating agents in    SSC-25 human squamous carcinoma cells. Cancer Chem and Pharmacol    1991, 29, 207-213.-   Schwartz J L, Antoniades D Z, Zhao S. Molecular and biochemical    reprogramming of oncogenesis through the activity of antioxidants    and prooxidants. Ann NY Acad Sci 1992, 686, 292-279.-   Schwartz J L, Flynn E A, Shklar G. The effect of carotenoids on    antitumor immune response in vivo and in vitro with hamster and    mouse immune effectors. Ann NY Acad Sci 1990, 587, 92-109.-   Schwartz J L, Shklar G, Trickler D. p53 in the anticancer mechanism    of vitamin E. Oral Oncol 1993, 29B, 313-183.-   Sies H, Stahl W. Vitamins E, C, β-carotene and other carotenoids as    antioxidants as antioxidants. Am. J. Clin. Nutr. 1995, 62,    1315-1321.-   Smith E M, Hoffman H T, Summersgill K S, Kirchner H L, Turek L P,    Haugen T H. Human papillomavirus and risk of oral cancer. Int J    Cancer 1998, 77, 341-346-   Stahl W, Junghans A, deBoer B, Driomina E S. et al. Caroteoid    mixtures protect multieamillar liposomes against oxidative damage;    synergistic effects of lycopene and lutein. FEBS Lett 1998, 427,    305-308.-   Steinberg D, et al. Antioxidants in the prevention of human    atheroscelrosis. Summary of the proceedings of a National Heart,    Lung and Blood Institute Workshop: Circulation 1992, 85, 2337-2344.-   Steinberg D, Parthasarathy S, Carew T E, Khoo J C and Witztum J L.    Beyond cholesterol: modifications of low-density lipoprotein that    increase its atherogenecity. N Engl. J. Med. 1989, 320, 915-924.-   Sthal W, Sies H. Uptake of lycopene and its geometrical isomers is    greater from heat-processed than form unprocessed tomato juice in    humans. J. Nutr. 1992, 122, 2161-2166.-   Stich H F, Roisin M P, Homby A P et al: Remission of oral    leukoplakias and micronuclei in tobacco/betel quid chewers treated    with beta-carotene and with beta-carotene plus vitamin A. Int J    Cancer 1998, 421, 195-199.-   Tanaka T, Morishita Y, Suzui M, Kojima T et al. Chemo prevention of    mouse urinary bladder carcinogenesis by the naturally occuring    carotenoid astaxanthin. Carcinogenesis. 1994, 15, 15-19.-   Wagner J R, Motchnik P A, Stocker R, Sies H, Ames B N. The oxidation    of blood plasma and low-density lipoprotein components by chemically    generated single oxygen. J. Biol. Chem. 1993, 268, 18502-18506.-   Watson A D, Navab M, Hama S Y, Sevanian A et al. Effect of platelet    activating factor-acetyl hydrolase on the formation and action of    minimally oxidized low-density lipoproteins. J. Clin. Invest. 1995,    95, 774-782.-   Weisburger J H. Mechanisms of action of antioxidants as exemplified    in vegetables, tomatoes and tea. Food Chem Toxicol 1999, 37,    943-948.-   Woodall A A, Lee S W, Wesie R J, Jackson M J and Britton G.    Oxidation of carotenoids by free radicals: relationship between    structure and reactivity. Biochim. Biophys. Acta 1997, 1336, 33-42.-   Yao 1, Iwai M, Furuta I. Correlation of bcl-2 and p53 expression    with clinicopathological features in tongue sqamous cell carcinomas.    Oral Oncol 1999, 35, 56-62.-   Yla-Herttuala S, Palinski W, Rosenfeld M E, Parthasarathy S. et al.    Evidance for the presence of oxidatively modified low-density    lipoprotein in atherosclerotic lesions of rabbit and mice. J. Clin.    Invest. 1989, 84, 1086-1095.-   Ziegler R G, A view of the epidemiological evidance that carotenoids    reduce the risk of cancer. J. Nutr. 1988, 119, 116-122.

1. A method of increasing a fraction of free carotenoids in a source ofcarotenoids in which at least some of the carotenoids are fatty acidesterified carotenoids, the method comprising contacting the source ofcarotenoids with an effective amount of an immobilized esterase underconditions effective in deesterifying the fatty acid esterifiedcarotenoids, thereby increasing the fraction of free carotenoids in thesource of carotenoids.
 2. The method of claim 1, wherein saidimmobilized esterase is selected from the group consisting of animmobilized lipase, an immobilized carboxyl ester esterase and animmobilized chlorophylase.
 3. The method of claim 1, wherein saidimmobilized esterase is an immobilized lipase.
 4. The method of claim 3,wherein said immobilized lipase is selected from the group consisting ofan immobilized bacterial lipase, immobilized yeast lipase, immobilizedmold lipase and immobilized animal lipase.
 5. The method of claim 3,wherein said immobilized lipase is a recycled immobilized lipase.
 6. Themethod of claim 1, wherein said source of carotenoids is characterizedin that a majority of the carotenoids in said source of carotenoids aresaid fatty acid esterified carotenoids.
 7. The method of claim 1,wherein said conditions effective in deesterifying the fatty acidesterified carotenoids are characterized by addition of at least oneadditive selected from the group consisting of: a cellulose degradingenzyme; a protein degrading enzyme; a pectin degrading enzyme; anemulsifier; and at least one metal ion.
 8. The method of claim 1,wherein said source of carotenoids is red pepper.
 9. The method of claim1, wherein said source of carotenoids is red pepper powder.
 10. Themethod of claim 1, wherein said source of carotenoids is paprika. 11.The method of claim 1, wherein said source of carotenoids is red pepperoil extract.
 12. The method of claim 1, wherein said source ofcarotenoids is red pepper oleoresin.
 13. The method of claim 1, whereinsaid source of carotenoids is selected from the group consisting ofapple, apricot, avocado, blood orange cape goosberry, carambola, chilli,clementine, kumquat, loquat, mango, minneola, nactarine, orange, papaya,peach, persimmon, plum, potato, pumpkin, tangerine and zucchini.
 14. Themethod of claim 7, wherein said at least one metal ion is selected fromthe group consisting of Ca⁺⁺ and Na⁺.
 15. The method of claim 7, whereinsaid addition of said at least one metal ion is by addition of at leastone salt of said metal ion.
 16. The method of claim 8, wherein said atleast one salt is selected from the group consisting of CaCl₂ and NaCl.17. The method of claim 7, wherein said cellulose degrading enzyme isselected from the group consisting of C1 type beta-1,4 glucanase,exo-beta-1,4 glucanase, endo-beta-1,4 glucanase and beta-glucosidase.18. The method of claim 7, wherein said proteins degrading enzyme isselected from the group consisting of trypsin, papain, chymotrypsins,ficin, bromelin, cathepsins and rennin.
 19. The method of claim 7,wherein said pectin degrading enzyme is selected from the groupconsisting of a pectin esterase, pectate lyase and a polygalacturonase.20. The method of claim 7, wherein said emulsifier is a non-esteremulsifier.
 21. The method of claim 7, wherein said emulsifier islecithin.
 22. The method of claim 20, wherein said emulsifier isdeoxycholate.
 23. The method of claim 7, wherein said emulsifier is anon-ionic detergent.
 24. The method of claim 20, wherein said emulsifieris derived from bile, gum Arabic or salt of free fatty acids.
 25. Themethod of claim 7, wherein said emulsifier is a recycled emulsifier. 26.The method of claim 1, further comprising extracting said source of atleast partially deesterified carotenoids with ethyl acetate underalkaline conditions.
 27. The method of claim 26, wherein said alkalineconditions are characterized by pH from about 8.0 to about
 10. 28. Themethod of claim 26, wherein said alkaline conditions are pH 9.5.